Glycosylated lysosomal proteins, method of production and uses

ABSTRACT

The present invention relates to a lysosomal protein composition comprising a plurality of lysosomal proteins that are potentially diversely glycosylated according to a glycosylation pattern, wherein said glycosylation pattern has at least 45% paucimannosidic N-glycans; a method of manufacturing the lysosomal protein composition in a bryophyte plant or cell, and medical and non-medical uses of the lysosomal protein composition. E.g. the lysosomal protein can be a-Galactosidase for the treatment of Fabry Disease or β-Glucoceramidase for the treatment of Gaucher&#39;s Disease. The unique glycosylation results in improved therapeutic efficacy—surprisingly even without mannose-6-phosphate that is common for CHO cell produced lysosomal proteins.

The present invention relates to the field of recombinant proteinexpression in plants for obtaining a modified glycosylation as comparedto mammalian expression systems.

BACKGROUND

Lysosomal storage diseases (LSDs) are a group of life-threateninginherited disorders; most of them are caused by deficiency of a singlelysosomal enzyme or protein, which leads to abnormal accumulation ofsubstrate in cells. Currently, enzyme replacement therapy (ERT) is theonly available specific treatment for several LSDs. In these diseases,lysosomal storage can be cleared in many target tissues by intravenousinfusion of the missing enzyme. Traditionally, recombinant enzymes usedin ERT are produced in cultured mammalian cells. E.g. U.S. Pat. No.6,083,725 describes an α-galactosidase from human cells. Recently, as analternative approach, plant-based expression systems have been utilizedto produce lysosomal enzymes for therapeutic use (Shaaltiel et al.(2007) Plant Biotechnol J 5:579-590; Du et al. (2008) J Lipid Res49:1646-1657; De Marchis et al. (2011) Plant Biotechnol J 9:1061-1073;He et al. (2012) Nat Commun 3:1062). Relative to mammalian cell-basedsystems, plant-based systems have several advantages including lowerproduction costs, eliminated risk of contamination by mammalianpathogens and, in the case of moss, a relatively easier manipulation ofthe N-glycosylation pathway. However, a major concern of plantcell-produced enzymes for ERT is their N-glycan structures that differfrom mammalian cell-produced enzymes. Particularly, lysosomal enzymesexpressed in plant cells typically do not acquire mannose 6-phosphate(M6P) modification on terminal mannose restudies without furtherartificial phosphorylation. Sugar chains exert a pivotal role in ERT.Intravenously administered lysosomal enzymes are taken up by tissuesthrough cell surface receptors that recognize carbohydrate structure ofthe enzymes. M6P receptor (M6PR) and mannose receptor (MR) represent twomajor contributors for this uptake system.

Most lysosomal enzymes carry M6P residues. It is generally believed thatin the ERT for most LSDs the M6PR-mediated endocytic pathway is crucialfor sufficient enzyme delivery (Sly et al. (2006) Proc Natl Acad Sci USA103:15172-15177; Sands et al. (2001) J Biol Chem 276:43160-43165). MR—onthe contrary—is present on macrophages and is believed to facilitateonly therapeutic effect of ERT aiming at enzyme substitution in thesecells.

WO 02/08404 and WO 2012/098537 describe the production of variouslysosomal enzymes in tobacco.

WO 03/073839 describes the production of lysosomal enzymes in plantseeds, especially in seeds of tobacco plants.

U.S. Pat. No. 7,011,831 describes the production of lysosomal enzymeswith complex N-glycan glycosylation in insect cells.

WO 2008/132743 describes the production of high mannose glycosylatedlysosomal enzymes in tobacco using an expression construct with ERsignal peptide and a vacuolar targeting signal.

EP 2 789 686 A1 describes the modification of plant glycosylationpathways to produce mammalian-type phosphorylated glycans.

US 2006/040353 describes transferring beta-galactose onto N-linkedglycans. Glycoproteins with mannose-6-phosphate are suggested fortreatment of lysosomal diseases.

Chiba et al. produced human α-gal A from yeast S. cerevisiae (Chiba etal. (2002) Glycobiology 12:821-828). In that case, M6P is covered byterminal mannose, and the removal of mannose residues by bacterialα-mannosidase led to improved M6PR-dependent uptake of the enzyme incultured fibroblasts. Recently, α-gal A was expressed in anothergene-manipulated yeast strain, which overexpresses MNN4, a positiveregulator of mannosylphosphate transferase (Tsukimura et al. (2012) MolMed 18:76-82). Phosphorylated N-glycan content in this α-gal Apreparation was higher than that in agalsidase alfa (28.7% vs. 15.3%).Repeated injection of this enzyme into Fabry mice resulted in similardecrease of cardiac and renal Gb₃ to that in agalsidase alfa-injectedmice. Most recently, Kizhner et al. reported the purification andcharacterization of human α-gal A produced from Tobacco cell culture(Kizhner et al. (2015) Mol Genet Metab 114(2): 259-267). As otherplant-made lysosomal enzymes, this aGal is non-phosphorylated. However,this protein is chemically cross-linked and PEGylated. Thesemodifications are associated with significant changes in proteincharacteristics including increased in vitro stability and dramaticallyprolonged circulation half-life (˜10 hr) when compared with agalsidasealfa or beta. The uptake mechanism of this enzyme remains to beelucidated. However, remarkably slow plasma clearance suggests that theuptake is not via M6PR- or MR-mediated endocytosis.

U.S. Pat. No. 6,887,696 describes a method for the expression of twolysosomal proteins, i.e. alpha-glucocerebrosidase andalpha-galactosidase, in tobacco plants, a higher plant. The tobaccoproduced lysosomal proteins had a diverse glycosylation pattern, havinghigh amounts of complex N-glycans, especially GnMXF, MGnXF, GnGnXF, GnMxand MGnX.

The goal of the invention is to provide an alternative source for theproduction of lysosomal proteins, which are active as therapeutics forthe treatment of lysosomal storage diseases requiring a suitableglycosylation.

SUMMARY OF THE INVENTION

This goal of the present invention is solved by the present invention,which is based on the surprising findings that i) paucimannosidicglycosylations on lysosomal proteins bestow suitability for treatmentoptions and ii) that such paucimannosidic glycosylations can be easilyobtained in bryophyte expression systems.

The present invention provides a method of manufacturing a lysosomalprotein composition comprising expressing a transgene encoding alysosomal protein in a bryophyte plant or cell, wherein said lysosomalprotein is expressed with a N-terminal secretory signal, wherein saidsecretory signal is optionally removed during intracellular processing,especially wherein the lysosomal protein lacks a C-terminal vacuolarsignal with the sequence VDTM (SEQ ID NO: 1), and said method furthercomprises obtaining an expressed lysosomal protein from said plant orcell. The invention further provides a lysosomal protein compositionobtainable by the inventive method.

The invention further provides a lysosomal protein compositioncomprising a plurality of lysosomal proteins that are potentiallydiversely glycosylated according to a glycosylation pattern, whereinsaid glycosylation pattern has at least 45% paucimannosidic N-glycans(molar %). Such a protein composition can be obtained by the inventivemethod.

Also provided a method of processing a lysosomal protein comprising acomplex N-glycan, said method comprising providing the lysosomal proteinin a sample and contacting the sample with a bryophyte HEXO, preferablyHEXO3, enzyme, whereby the bryophyte HEXO enzyme cleaves terminal GlcNAcresidues from the lysosomal protein thereby producing a paucimannosidicN-glycan. The HEXO enzyme may be in a cell, especially a plant cell. Thelysosomal proteins can be provided as a medicament or in apharmaceutical composition.

The invention also provides a bryophyte cell or plant suitable forperforming the inventive method comprising said transgene encoding alysosomal protein.

The invention also relates to a method of treatment of a lysosomalstorage disease comprising administering a lysosomal protein compositionaccording to the invention to a patient in need of treatment.

All these aspects are interrelated, equally form part of the entireinvention presented herein and preferred embodiments of the invention inany combination may relate to any one of these aspects, e.g. plants orcells transformed by a given construct or method can be provided or usedin the production of any lysosomal protein to be glycosylated accordingto the invention. The following detailed description on any embodimentor preferred feature relates to all aspects equally. E.g. a productfeature of the lysosomal protein means that the method is selected toproduce this lysosomal protein with its product feature. A descriptionof particular method steps is equally descriptive of the proteinmodified by this method step. The inventive cell is transformed tofacilitate any inventive method of manufacture in the cell. Alllysosomal proteins can be used in the inventive methods of(therapeutically or non-therapeutically) using the lysosomal protein.The present invention is further defined in the claims.

DETAILED DESCRIPTION OF THE INVENTION

Lysosomal storage diseases (LSD) are a group of approximately 50 rareinherited metabolic disorders that result from defects in lysosomalfunction. Lysosomes are responsible for digesting various moleculesinvolving several critical enzymes. If one of these enzymes isdefective, because of a mutation, the large molecules accumulate withinthe cell, eventually killing it. Lysosomal storage disorders are causedby lysosomal dysfunction usually as a consequence of deficiency of asingle enzyme required for the metabolism of lipids, glycoproteins ormucopolysaccharides.

Treatment of lysosomal storage diseases is mostly symptomatic, withenzyme replacement therapy being the most common. ERT requiresadministration of active lysosomal proteins into the cells via an uptakeroute.

Traditionally, recombinant enzymes used in ERT are produced in culturedmammalian cells. Recently, as an alternative approach, plant-basedexpression systems have been utilized to produce lysosomal enzymes fortherapeutic use. Relative to mammalian cell-based systems, plant-basedsystems have several advantages including lower production costs,eliminated risk of contamination by mammalian pathogens and, in the caseof moss, a relatively easier manipulation of the N-glycosylationpathway. However, a major concern when considering using plantcell-produced enzymes for ERT is their N-glycan structures that differfrom mammalian cell-produced enzymes. Particularly, lysosomal enzymesexpressed in plant cells typically do not acquire mannose 6-phosphate(M6P) modification on terminal mannose residues.

The present invention provides a new method of producing transgeniclysosomal proteins in plant cells, especially in bryophyte cells, whichsurprisingly led to the formation of glycoproteins with a high degree ofpaucimannosidic glycosylation, i.e. a glycosylation terminating withfew, e.g. 2 mannose residues, in a branched -Man<(Man)₂ structure. Thesestructures have proven to be highly effective for the uptake, especiallyin cells affected by lysosomal storage disease. This alteredglycosylation is unnatural for the lysosomal proteins, yet surprisinglythe altered proteins are still very effective therapeutic proteins.

The lysosomal protein used in the inventive method is a transgene, e.g.of mammalian origin, for use in ERT in that mammal of origin. Thesetransgenic lysosomal proteins produced in bryophytes can be used astherapeutic proteins for the treatment of lysosomal storage diseases. Assuch, the lysosomal proteins are active enzymes when administered, inparticular active in conditions occurring in a lysosome, such as a pH ofabout 5. Of course inactive storage forms, e.g. when lyophilized, arepossible. The inventive paucimannosidic glycosylation does notsubstantially interfere with enzymatic activity but mediates uptake andstability of the lysosomal proteins. Surprisingly, the inventiveN-glycans on the lysosomal proteins lead to high efficacy allowing atherapeutic use thereof especially in the treatment of lysosomal storagediseases.

The present invention can rely on known methods for introducingtransgenes into bryophytes. Suitable transformation systems have beendeveloped for the biotechnological exploitation of bryophytes for theproduction of heterologous proteins. For example, successfultransformations have been carried out by direct DNA transfer intoprotonema tissue using particle guns. PEG-mediated DNA transfer intomoss protoplasts has also been successfully achieved. The PEG-mediatedtransformation method has been described many times for Physcomitrellapatens and leads both to transient and to stable transformants (K.Reutter and R. Reski, Pl. Tissue culture and Biotech., 2, 142-147(1996)). Moreover, marker-free transformation can be achieved byPEG-mediated transformation method with bryophytes as well (Stemmer C,Koch A and Gorr G (2004), Moss 2004, The 7th Annual Moss InternationalConference, Freiburg, Germany) and can be used for subsequentintroduction of multiple nucleotide sequences.

Detailed information on the culturing of bryophytes which are suitablefor use in the invention, such as Leptobryum pyriforme and Sphagnummagellanicum in bioreactors, is known in the prior art (E. Wilbert,“Biotechnological studies concerning the mass culture of mosses withparticular consideration of the arachidonic acid metabolism”, Ph.D.thesis, University of Mainz (1991); H. Rudolph and S. Rasmussen, Crypt.Bot., 3, 67-73 (1992)). Especially preferred for the purposes of thepresent invention is the use of Physcomitrella patens, since molecularbiology techniques are practiced on this organism (R. Reski Bot. Acta,111, pp. 1-15 (1998)). For cultivation of bryophytes media with (Baur etal. (2005) Plant Biotechnol J 3, 331-340) or without supplements liketrace elements can be used (Weise et al. (2006) Appl. Microbiol.Biotechnol., 70, 337-345).

The inventive method of manufacturing a lysosomal protein compositionpreferably comprises expressing a transgene encoding a lysosomal proteinin a bryophyte plant or cell, wherein said lysosomal protein isexpressed with a N-terminal secretory signal and the lysosomal proteinlacks a C-terminal vacuolar signal with the sequence VDTM (SEQ ID NO:1). This would avoid vacuolar targeting, which would lead to storage ofthe lysosomal protein in the vacuole (contrary to excretion) andpotentially to different glycol-processing in the vacuole, wherein stillpaucimannosidic glycoforms are still possible to some extent. In thegolgi, without vacuole targeting, based on bryophyte specificrecombinant protein interaction, a different glycosylation pathwaysurprisingly led to high amounts of paucimannosidic glycosylationindependent of vacuole processing. This is very surprising since intobacco the secretory, non-vacuole pathway led to the formation ofpredominantly complex N-glycans instead of paucimannosidic glycosylation(U.S. Pat. No. 6,887,696). Apparently, bryophytes have a uniquerecognition of lysosomal proteins leading to this modification.

SEQ ID NO: 1 is a C-terminal plant vacuolar targeting signal leading toefficient vacuole targeting. In some embodiments, other vacuoletargeting signals may be present, especially non-plant signals, leadingto less efficient vacuole targeting and some expression down thesecretory pathway avoiding the vacuole. However, in most preferredembodiments, no vacuole targeting signal is present during expression oreven in the final obtained product. Vacuolar signals may also be removedin artificially after obtaining the protein from the cells.

Paucimannosidic N-glycans are based on trimming of complex N-glycans. Inthe golgi, the terminal GlcNAc is removed from complex glycans leaving aterminal mannose, in case of the bryophyte system, this is veryefficient leaving terminal mannose on both branches of the (formerlycomplex) N-glycan.

According to the invention, a secretory signal sequence is used, usuallyon the N-terminus of the amino acid sequence. The N-terminal secretorysignal is also referred to as a transit peptide or ER signal sequence.It is part of the encoded and expressed amino acid sequence. Thesecretory signal leads to an expression directly into the ER of a cell,setting the pathways for secretion (or to vacuolar designation if avascular signal is present. The secretory signal is usually removedintrinsically from the protein amino acid sequence during expression.This is a natural process in a plant cell. To allow proper localizationof the expression product of the transgenes, the genes for the lysosomalproteins can be modified to allow for localization in the plant cell.Preferably hybrid nucleic acid sequences are used in the constructs forthe transformation of the plants or plant cells. Localization-relevantdomains of the e.g. mammalian enzymes are replaced by plant sequences toachieve correct localization and cellular transit such as in the ERand/or golgi in planta. An example of a plant secretory signal is SEQ IDNO: 5, but any other plant secretory signal may be used. It may be anendogenous sequence to the used bryophyte species or it may be a foreignplant sequence, but preferably still a bryophyte sequence.

The inventive method includes expression of the lysosomal proteinwithout a plant (bryophyte) vacuolar signal, which has the sequence VDTM(SEQ ID NO: 1). This leads to an expression pathway from the ER to thegolgi and eventually to secretion, avoiding the end-localization in avacuole. In bryophytes, the secretion can be directly into the culturingmedium. In other plants it may be to an apoplastic compartment of theplant cell. Surprisingly, even without vacuole placement, a high degreeof paucomannosidic glycosylation could be achieved by the inventivemethod.

As a final step, the expressed lysosomal protein from said plant or cellis then obtained. To this end, the lysosomal protein may be collectedfrom an extraction process from the cells, which may be disruptive ornon-disruptive. Preferably the expressed lysosomal protein is obtainedfrom secreted matter of the plant or cell, e.g. from the culture medium,preferably without disrupting the producing cells or plant. The obtainedlysosomal protein may then be purified, e.g. to a concentration of atleast 80% (m/v), preferably at least 90% (m/v), especially preferred atleast 95% (m/v), or at least 98% (m/v) or at least 99% (m/v).

Preferably the bryophyte plant or cell is a moss, preferably P. patens,plant or cell. The bryophyte may be any bryophyte but is preferablyselected from moss, liverwort or hornwort, especially preferred of theclass bryopsida or of the genera Physcomitrella, Funaria, Sphagnum,Ceratodon, Marchantia and Sphaerocarpos. Physcomitrella patens is aparticularly preferred system as it has a high rate of homologousrecombination.

Subject matter of the invention are plants and plant cells. “Plant cell”as used herein may refer to an isolated cell, a singularized cell, butalso a cell in or of a plant tissue, preferably a tissue selected fromcallus, protonema, phloem, xylem, mesophyll, trunk, leaves, thallus,chloronema, caulonema, rhizoid or gametophore, or a cell in a plantorganism. The cell may be a protoplast cell. In preferred embodiments,isolated plant cells or even plant tissues are transformed according tothe invention and then grown into plants or plant tissues, or remainplant cell cultures, such as a suspension culture, e.g. a bioreactor(Hobe & Reski, Plant Cell, Tissue and Organ Culture 81, 2005: 307-311).

Preferably the lysosomal protein further lacks a C-terminal ER retentionsignal with the sequence KDEL (SEQ ID NO: 2). ER retention signals leadto a retention in the ER or golgi system. This can have a profoundimpact on the glycosylation pattern observed in the expressed proteinsince glycosylation is a competitive process with several glycosylatingenzymes vying for the substrate proteins to be modified. Although onecould have expected that paucimannosidic trimming would benefit from ERretention, surprisingly the inventive lysosomal proteins were expressedwith high amounts of paucimannosidic N-glycans without this (or any) ERretention signal.

SEQ ID NO: 2 is a C-terminal ER retention signal leading to efficient ERor golgi retention. Also, preferred the C-terminal di-lysine motif(KXKX), also responsible for ER retention to some extent, is alsomissing. In some embodiments, other ER retention signals may be present,especially non-plant signals, leading to less efficient ER/golgiretention and faster processing from compartment to compartment towardssecretion. However, in most preferred embodiments, no ER retentionsignal is present during expression or even in the final obtainedproduct. ER retention signals may also be removed artificially afterobtaining the protein from the cells.

Especially preferred for any embodiment of the present invention, thelysosomal protein lacks a plant C-terminal ER retention signal sequenceand a plant C-terminal vacuolar signal sequence, especially preferred,it lacks any C-terminal ER retention signal sequence and any C-terminalvacuolar signal sequence. Plant signals are from plant origin and foundin plants, especially bryophytes. They are functional in bryophytes. Asexplained above, ER retention is not necessary and even vacuolarprocessing is not required for the high paucimannosidic glycosylation inthe inventive bryophyte expression systems.

Preferably the lysosomal protein comprises an expressed amino acidsequence that terminates on the C-terminus with the amino acids of anative lysosomal protein or a truncation thereof This means that noadditions to the proteins sequence are present. Truncations arepossible, even if not preferred. Of course the truncations do notsubstantially affect activity of the inventive lysosomal protein, thatis still a requirement as explained above. Enzymatic activity may bereduced by e.g. up to 20% when compared to the native lysosomal proteinin lysosomal conditions in a mammalian, especially human, cell, such asat a pH of 5. The truncations may be a deletion of at most 50,preferably at most 40, at most 30, at most 20, at most 10, at most 5 orone, or any range in between these values (e.g. 1 to 10 etc.) C-terminalamino acids of the native lysosomal protein. Truncatedalpha-galactosidases are known to be active with such truncations ase.g. described in U.S. Pat. No. 6,887,696 B2 (incorporated herein byreference).

Preferably the bryophyte plant or cell does or does not comprise a HEXO3transgene. HEXO3 is an enzyme that is found naturally in plants. It maybe supplied (or not) as a transgene to even further increase HEXO3activity. Introductions of transgenes may be facilitated by the samemethods as the lysosomal protein transgene is incorporated into theplant or cell, e.g. by genomic recombinant hybridization or plasmidintroduction. HEXO3 is said to be responsible for some pauimannosidicglycosylation in the apoplast lining plasma membrane of plants(Liebminger et al., J Biol Chem 2011, 286: 10793-10802; Bosch et al.,Curr Pharm Des. 2013; 19(31):5503-12), however the HEXO activity foundby Liebminger in Arabidopsis thaliana cannot explain the highpaucimannosidic glycosylation found in bryophytes. Related enzyme HEXO1is responsible for some vacuolar paucimannosidic glycosylation and HEXO2seems to have little activity in Arabidopsis. There can be higheractivity or better accessibility in bryophytes.

Apparently bryophyte HEXO is particularly highly active on lysosomalproteins. Therefore the present invention provides an in vitro method ofprocessing a lysosomal protein comprising a complex N-glycan, saidmethod comprising providing the lysosomal protein in a sample andcontacting the sample with a bryophyte HEXO, preferably HEXO3, enzyme,whereby the bryophyte HEXO enzyme cleaves terminal GlcNAc residues fromthe lysosomal protein thereby producing a paucimannosidic N-glycan. Inessence, the native lysosomal protein glycosylation pathway found inbryophytes can be also performed outside a bryophyte cell, especially invitro with isolated HEXO enzymes, or in another cell, preferably plantcell by substituting that plant with an active HEXO enzyme from abryophyte, especially a moss such as P. patens. This plant may be anon-bryophyte, e.g. a higher plant, or a bryophyte to increase HEXO,preferably HEXO3, gene load as detailed above.

Preferably the bryophyte plant or cell has suppressed or eliminatedalpha1,3-fucosyltransferase and/or beta1,2-xylosyltransferase. Suchplant enzymes can be reduced in activity or concentration, at least inthe site of their natural activity such as the golgi. Enzymes that arepreferably removed are alpha-1,3-fucosyltransferase and/orbeta-1,2-xylosyltransferase as described in WO 2004/057002. Thusaccording to a preferred embodiment, said plant cell further has areduced activity, preferably a complete loss of function, ofalpha-1,3-fucosyltransferase and/or of beta-1,2-xylosyltransferase, inparticular by knock-out, especially preferred by interrupting thealpha-1,3-fucosyltransferase and/or beta-1,2-xylosyltransferase encodinggene of said plant, preferably by a gene of any one of the recombinantlyexpressed proteins. This measure prevents formation of plant-typeglycosylations that may be immunogenic in a mammal such as a human.

Also provided is a bryophyte cell or plant suitable for performing thismethod. The cell or plant comprises a transgene encoding a lysosomalprotein as described for the method paratively and optionally anyfurther modification or transgene as described above.

The present invention further provides a lysosomal protein compositionobtainable by any method of manufacturing described herein. Thelysosomal protein may have any characteristics as effected by a methodor preferred variant or embodiment as described above. The lysosomalprotein is usually obtained in a plurality of such lysosomal proteins,with the inventive lysosomal glycosylation pattern observed inbryophytes for (transgenic) lysosomal proteins.

Especially, the invention provides a lysosomal protein compositioncomprising a plurality of lysosomal proteins that are potentiallydiversely glycosylated according to a glycosylation pattern, whereinsaid glycosylation pattern has at least 45%, preferably at least 50%, atleast 55%, at least 60%, at least 65%, or at least 70%, paucimannosidicN-glycans.

All percentage values given herein are molar percentages, except whereindicated otherwise.

A plurality relates to preparation of the inventive proteins comprising,i.e. not individualized proteins but a macroscopic preparation of suchproteins as obtained from the cells or plants, which comprises more thanone lysosomal protein when expressed. The preparation may have at least1000 protein molecules, especially preferred at least 100000 moleculesor at least 1 million molecules.

Preferably the lysosomal protein of the composition or produced or usedby or in the inventive methods is any one selected from α-Galactosidase,preferably α-Galactosidase A (GLA); β-Glucoceramidase, β-glucosidase(glucocerebrosidase); α-Mannosidase; Aspartylglucosaminidase;β-Mannosidase; Acid Ceremidase; α-Fucosidase; β-Galactosidase,β-Hexosaminidase activator protein; Galactocerebrosidase,Galactoceramidase; lysosomal acid lipase (LAL); α-Iduronidase;Iduronate-2-sulfatase; Glucosamine-N-sulfatase, Heparansulfatsulfamidase(SGSH); α-N-acetyl-glucosaminidase (NAGLU);(Heparan)α-glucosaminide-N-acetyltransferase;N-Acetygalactosamine-6-sulfatase; β-Galactosidase;N-Acetygalactosamine-4-sulfatase; β-Glucoronidase; Neuraminidase;Sphingomyelinase, Sphingomyelin phosphodiesterase; Acidalpha-1,4-glucosidase; β-Hexosaminidase, or its α subunit;Alpha-N-acetylgalactosaminidase (NAGA), α-Galactosaminidase;β-Hexosaminidase A; Galactose-6-sulfate sulfatase; Hyaluronidase.Especially preferred in all embodiments of the invention isα-Galactosidase. A preferred α-Galactosidase is human α-Galactosidase,e.g. of SEQ ID NO: 3, which can be expressed from the nucleic acidsequence of SEQ ID NO: 4, or a truncated α-Galactosidase therefrom. Alsopreferred is glucocerebrosidase, e.g. human glucocerebrosidase e.g. ofSEQ ID NO: 6, which can be expressed from the nucleic acid sequence ofSEQ ID NO: 7. Also preferred is alpha-glucosidae, e.g. humanalpha-glucosidae e.g. of SEQ ID NO: 8, which can be expressed from thenucleic acid sequence of SEQ ID NO: 9. In other embodiments,glucocerebrosidase and alpha-glucosidase are excluded from the group oflysosomal proteins according to the invention.

Preferably the lysosomal proteins have one or more paucimannosidicN-glycans comprising the structure of formula 1:

wherein a square represents N-Acetylglucosamine (GlcNAc), a circlerepresents mannose (Man), and a circle with a T represents a terminalmannose. This formula 1, also referred herein as “MM” glycan, representsa core structure that may be further modified—in paucimannosidicN-glycans this further modification is also possible as long as theT-mannoses remain terminal, i.e. are at the non-reducing ends of thesugar chains. The terminal mannoses may be methylated, especiallyO-methylated. Common modifications are where one or more of the GlcNAcor Man subunits may be α(1,3-fucosylated, α1,6-fucosylated and/orβ1,2-xylosylated. α(1,3-fucosylations and α1,6-fucosylatations are foundcommonly on the reducing GlcNAc. A β1,2-xylosylation is usually found atthe non-terminal mannose (circle without T in formula 1). According tothe invention, preferably a α1,3-fucosylation and/or β1,2-xylosylationis prevented or reduced due to the inhibition of the respective enzymesduring manufacture as mentioned above. α1,6-fucosylation may or may notbe present. It is uncommon in plants but may be achieved by introductionof a α1,6-fucosyltransferase into the expressing cell or plant.Preferably at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, or at least 95%, of the N-glycans of the lysosomal proteinsof the inventive compositions comprise or consist of the structure offormula 1 (molar %).

A paucimannosidic N-glycan structure of the invention can also berepresented by formula (2):

Formula 2 further shows the type of carbohydrate subunit connectivity.The GlcNAc to the right is bound to the amino acid sequence of thelysosomal protein. The reducing and non-reducing ends of anoligosaccharide are conventionally drawn with the reducing-endmonosaccharide residue furthest to the right and the non-reducing endfurthest to the left (as e.g. in formula 2). Note, the reducing GlcNAcis shown left in the short formulas given herein, such as -GlcNAc₂-Man₃.In an N-glycan, the reducing -GlcNAc is bound to an Asparagine in theamino acid chain of the lysosomal protein. This is indicated by the left“-” being a bond to the Asn residue. In paucimannosidic glycoforms, twonon-reducing mannose termini exist (left in formula 2, up in formula 1).

Formula (2) is the common core of most N-glycans, including high-mannoseand complex N-glycans (see Rayon et al., J Exp. Botany, 1998, 49(326):1463-1472). In case of paucimannosidic structures, both upper and lowerMan to the left as shown in formula (2) are terminal, whereas in highmannose and complex N-glycans at least one Man contains a further bondto another Man or GlcNAc. α1,3-Fucosylation, α1,6-fucosylation and/orβ1,2-xylosylation of this core are optional. α1,3-Fucosylation andβ1,2-xylosylation are common in plants unless the respective enzymes areinhibited (e.g. as shown in WO 2004/057002 or Cox et al., NatureBiotechnology 24(12), 2006: 1591-7).

Should the expressed lysosomal proteins have amounts of MGn glycans (oneadditional terminal N-acetylglucosamine at one terminal Man as shown informula 2) or GG glycans (one additional terminal N-acetylglucosamine ateach terminal Man as shown in formula 2), these MGn and GG glycans canbe converted to the structures of formula 1 or 2 by treatment withbeta-N-Acetylglucosaminidase. Beta-N-Acetylglucosaminidase treatment canbe performed with any moss produced lyososmal protein to increasepaucimannosidic glycans. Moss expressed α-Galactosidase A usually doesnot need beta-N-Acetylglucosaminidase since paucimannosidic glycans arenaturally in high concentrations. Depending on culturing conditions,moss produced glucocerebrosidase and alpha-glucosidase may sometimesneed beta-N-Acetylglucosaminidase treatment. Other methods to create theinventive glycosylated lysosomal proteins include expression in insectsor insect cells, such as Sf9 cells, expression in glyco engineered yeastcells and expression in moss with a vacuolar targeting signal (althoughless preferred since the signal may be immunogenic in a mammalianpatient). Any description herein of “moss produced” or “bryophyteproduced” lysosomal proteins relates to the products obtainable by theproduction in moss, i.e. having the inventive paucimannosidic N-glycansin high amounts as described herein, irrespective of the actual methodof manufacture. Any method of manufacture can be selected to produce theinventive products and “moss produced” or “bryophyte produced” alsorefers to these products on non-moss production methods. “Moss produced”or “bryophyte produced” is used to express the unique glycosylationpattern, that was found in moss and is defined herein particularly, e.g.in product claims and the description of such products herein.

A unique characteristic of the bryophyte N-glycosylation of thelysosomal proteins according to the inventive method is the presence ofmethylated hexoses (Hex), preferably methylated mannose (Man) in theinventive N-glycans. These methylated mannoses, if terminal, do notinterfere with uptake of the inventive lysosomal proteins and can evenenhance it. Preferably the glycosylation pattern of the inventivecomposition has at least 1%, more preferred at least 2%, at least 3%, orat least 4%, N-glycans of the formula GlcNAc₂-Hex₂-methyl-Hex, with Hexbeing preferably mannose (molar %). According to the inventive method,the methylation is usually a methylation of an oxygen of mannose, inparticular a 2-O-methylation. These are preferred methylations of theinventive lysosomal proteins of the composition. Preferably, at least1%, more preferred at least 2%, at least 3%, or at least 4%, of theN-glycans of the composition have a methylation, especially a O—, e.g.2-O-methylation (molar %). Preferably in these methylated N-glycans,only one of the at least two terminal (non-reducing) mannoses ismethylated.

Preferably the glycosylation pattern comprises the following N-glycans:

i) 0% to 35%, preferably 0.5% to 30%, -GlcNAc₂-(Man₂methyl-Hex);

ii) 30% to 80%, preferably 40% to 70%, especially preferred 45% to 60%,-GlcNAc₂-Man₃;

iii) 0% to 30%, preferably 4% to 22%, -GlcNAc₂-Man₃-GlcNAc;

iv) 0% to 15%, preferably 2% to 12%, -GlcNAc₂-Man₃-GlcNAc₂;

v) 0% to 5%, preferably 0% to 3%, -GlcNAc₂-Man₃-Hex₂;

vi) 0% to 11%, preferably 1% to 8%, -GlcNAc₂-Man₃-Hex₃;

vii) 0% to 10%, preferably 1% to 7%, -GlcNAc₂-Man₃-Hex₄;

viii) 0% to 10%, preferably 1% to 7%, -GlcNAc₂-Man₃-Hex₅;

wherein all of these compounds together amount to 100% or less than100%, which is self-evident (all % are molar %). Less than 100% arepossible since other N-glycans, not specified in the list above may bepresent. Such other N-glycans may be between 0% and 30%, preferablybetween 0.01% and 20%, especially preferred between 0.1% and 10%. Anyone of the specified N-glycans i) to viii) may be in an amount of atleast 0.01% instead of 0%. GlcNAc is a N-Acetylglucosamine subunit, Manis a mannose subunit, Hex is a hexose subunit, methyl-Hex is amethylated hexose subunit, preferably 2-O methyl hexose. In thisglycosylation pattern -GlcNAc₂-(Man₂methyl-Hex) and -GlcNAc₂-Man₃together amount to at least 45% (molar %), i.e. these arepaucimannosidic N-glycans that contribute to this amount as mentioned inthe summary of the invention. Especially preferred Hex is Man in any oneof the above N-glycans i) to viii). The GlcNAc at the reducing end ofthe glycan may be fucosylated or is not fucosylated in any one of theabove N-glycans i) to viii). A Man at a branching point is xylosylatedor is not xylosylated in any one of the above N-glycans i) to viii).

In a particular preferred embodiment, all N-glycans listed above i) toviii) are in the preferred amount as given in the preceding paragraph.

Also preferred, the glycosylation pattern comprises N-glycan i),-GlcNAc₂-(Man₂methyl-Hex), in an amount of at least 0.5%, at least 1%,at least 2% or at least 3%. Especially preferred, it is in an amount ofat most 30%, at most 25%, at most 20% or at most 15%. Its amount may bein the range 0.5% to 30%, 1% to 25% or 2% to 20%.

Also preferred, the glycosylation pattern comprises N-glycan ii),-GlcNAc₂-Man₃, in an amount of at least 30%, at least 40%, at least 45%or at least 50%. Especially preferred, it is in an amount of at most80%, at most 75%, at most 70% or at most 65%. Its amount may be in therange 30% to 80%, 40% to 70% or 45% to 60%. This is the most importantN-glycan according to the invention and may be present in these amountsin any embodiment of the invention.

Also preferred, the glycosylation pattern comprises N-glycan iii),-GlcNAc₂-Man₃-GlcNAc, in an amount of at least 0.5%, at least 2%, atleast 4% or at least 6%. Especially preferred, it is in an amount of atmost 30%, at most 25%, at most 20% or at most 15%. Its amount may be inthe range 0.5% to 30%, 1% to 25% or 2% to 20%.

Also preferred, the glycosylation pattern comprises N-glycan iv),-GlcNAc₂-Man₃-GlcNAc₂, in an amount of at least 0.2%, at least 0.5%, atleast 1% or at least 2%. Especially preferred, it is in an amount of atmost 20%, at most 15%, at most 12% or at most 10%. Its amount may be inthe range 0.5% to 15%, 1% to 12.5% or 2% to 10%.

Also preferred, the glycosylation pattern comprises N-glycan v),-GlcNAc₂-Man₃-Hex₂, in an amount of at least 0.01%, at least 0.05%, atleast 0.1% or at least 0.5%. Especially preferred, it is in an amount ofat most 5%, at most 4%, at most 3% or at most 2%. Its amount may be inthe range 0.1% to 5%, 0.2% to 4% or 0.5% to 3%.

Also preferred, the glycosylation pattern comprises N-glycan vi),-GlcNAc₂-Man₃-Hex₃, in an amount of at least 0.1%, at least 0.2%, atleast 0.75% or at least 1%. Especially preferred, it is in an amount ofat most 11%, at most 10%, at most 8% or at most 6%. Its amount may be inthe range 0.5% to 11%, 1% to 10% or 2% to 9%.

Also preferred, the glycosylation pattern comprises N-glycan vii),-GlcNAc₂-Man₃-Hex₄, in an amount of at least 0.1%, at least 0.2%, atleast 0.3% or at least 0.4%. Especially preferred, it is in an amount ofat most 10%, at most 8%, at most 6.5% or at most 5%. Its amount may bein the range 0.1% to 10%, 0.2% to 8.5% or 0.3% to 7%.

Also preferred, the glycosylation pattern comprises N-glycan viii),-GlcNAc₂-Man₃-Hex₅, in an amount of at least 0.1%, at least 0.2%, atleast 0.3% or at least 0.4%. Especially preferred, it is in an amount ofat most 10%, at most 8%, at most 6.5% or at most 5%. Its amount may bein the range 0.1% to 10%, 0.2% to 8.5% or 0.3% to 7%.

The inventive lysosomal proteins can be of any source, preferablymammalian, especially human or a non-human animal, such as a rodent, adog, cat, horse, cow, camel, pig.

Plant produced glycoproteins, including the inventive bryophyte producedlysosomal proteins are usually not mannose phosphorylated. Alsoaccording to the invention, the N-glycans may be non-mannosephosphorylated, especially not phosphorylated at all. Phosphorylationcan be done artificially by introducing a phosphorylating enzyme intothe expressing cell or by phosphorylation after isolation of the proteinfrom the cells. Thus, the lysosomal protein composition according to theinvention comprises non-phosphorylated or phosphorylated lysosomalproteins. Preferably the amount of phosphorylated N-glycans of thelysosomal proteins of the composition is below 20%, especially preferredbelow 15%, below 10%, below 5%, below 2%, below 1%, or even below 0.5%,e.g. 0% (molar %).

The inventive lysosomal proteins may be PEGylated non-PEGylated.PEGylation may modify the solubility, bioavailability and in vivohalf-life when administered to a patient. Given that half-life of theinventive paucimannosidicly glycosylated lysosomal proteins is reduceddue to smaller N-glycan structure as compared to mammalian producedlysosomal proteins, PEGylation is especially preferred to compensate forthis draw-back. Especially preferred is a reversible PEGylation, leadingto a at least partial loss of the PEGylation in vivo, e.g. byintroducing a hydrolysable bond, such as a Schiff base, so as not tointerfere with cellular uptake. Also as a measure to reduce cellularuptake interference, the PEGylation can be of short PEG chains, such asPEG with 4 to 1000, preferably 8 to 100 or 10 to 50 subunits. Instead ofPEG, any short hydrophilic polymer can be used to increase half-life,preferably with a molecular weight of less than 100 kDa, less than 10kDa or less than 1 kDa. Preferably PEG has 2-200, preferably 3 to 100 or4 to 50, glycol subunits. The lysosomatic protein can be PEGylated via alinking moiety as means for indirect attachment of the PEG molecule.Alternatively, also direct attachment is possible.

Preferably the inventive lysosomal proteins are non-crosslinked.Crosslinking can interfere with cellular uptake and stability and isless preferred. Preferably crosslinking is combined with PEGylation. Forexample, PEG can be used as cross-linking agent, especially in case ofbi- or multi-functional PEG having at least two functional groups forbinding to a protein, such as bis-COOH-PEG or bis-NHS-PEG. PEGylationcan mask the negative aspects of crosslinking that cause interferencewith cellular uptake and stability.

Crosslinking can lead to the formation of multimeric lysosomal proteins,especially preferred dimeric lysosomal proteins, as e.g. described foralpha-galactosidase is WO 2011/107990 (incorporated herein byreference). In cross-linked proteins, at least two lysosomal proteinsare connected either directly or indirectly via a linking moiety.

Cross-linking and/or PEGylation can be by linking moiety. for example,the linking moiety is optionally a moiety which is covalently attachedto a side chain, an N-terminus or a C-terminus, or a moiety related topost-translational modifications (e.g., a saccharide moiety) of anlysosomal protein monomer, as well as to a side chain, an N-terminus ora C-terminus, or a moiety related to post-translational modifications(e.g., a saccharide moiety) of another lysosomal protein monomer.Exemplary such linking moieties are described in detail hereinunder.

Alternatively, the linking moiety forms a part of the lysosomal proteinmonomers being linked (e.g., a part of a side chain, N-terminus orC-terminus, or a moiety related to post-translational modifications(e.g., a saccharide moiety) of an lysosomal protein monomer, as well asof a side chain, an N-terminus or a C-terminus, or a moiety related topost-translational modifications (e.g., a saccharide moiety) of anotherlysosomal protein monomer). Thus, for example, the linking moiety can bea covalent bond (e.g., an amide bond) between a functional group of aside chain, N-terminus, C-terminus or moiety related topost-translational modifications of a monomer (e.g., an amine), and acomplementary functional group of a side chain, N-terminus, C-terminusor moiety related to post-translational modifications of another monomer(e.g., carboxyl), wherein such a covalent bond is absent from the nativeform of the α-galactosidase. Other covalent bonds, such as, for example,an ester bond (between a hydroxy group and a carboxyl); a thioesterbond; an ether bond (between two hydroxy groups); a thioether bond; ananhydride bond (between two carboxyls); a thioamide bond; a carbamate orthiocarbamate bond, are also contemplated. Optionally, the linkingmoiety is devoid of a disulfide bond. However, a linking moiety whichincludes a disulfide bond at a position which does not form a linkbetween monomers (e.g., cleavage of the disulfide bond does not cleavethe link between the monomers) is within the scope of this embodiment ofthe invention. A potential advantage of linking moiety devoid of adisulfide bond is that it is not susceptible to cleavage by mildlyreducing conditions, as are disulfide bonds. Optionally, the linkingmoiety is a non-peptidic moiety (e.g., the linking moiety does notconsist of an amide bond, an amino acid, a dipeptide, a tripeptide, anoligopeptide or a polypeptide). Alternatively, the linking moiety maybe, or may comprise, a peptidic moiety (e.g., an amino acid, adipeptide, a tripeptide, an oligopeptide or a polypeptide). Optionally,the linking moiety is not merely a linear extension of any of thelysosomal protein monomers attached thereto (i.e., the N-terminus andC-terminus of the peptidic moiety is not attached directly to theC-terminus or N-terminus of any of the lysosomal protein monomers).Alternatively, the linking moiety is formed by direct covalentattachment of an N-terminus of a lysosomal protein monomer with aC-terminus of another lysosomal protein monomer, so as to produce afused polypeptide. Such a polypeptide will not be a native form ofα-galactosidase, although it may comprise two lysosomal protein monomersessentially in their native form. However, the covalent linking ofα-galactosidase monomers described herein is preferably in a form otherthan direct linkage of an N-terminus to a C-terminus. The linking moietyis preferably a small moiety of 10 to 1000 Da, preferably 20 to 500 Da.

In cross-linking and/or PEGylation, the linking moiety may comprise oneor more reactive group for binding to the lysosomal protein. Suchreactive groups may react for example with a thiol group or react withan amine group to form an amide bond. As used herein, the phrase“reactive group” describes a chemical group that is capable ofundergoing a chemical reaction that typically leads to a bond formation.The bond, according to the present embodiments, is preferably a covalentbond (e.g., for each of the reactive groups). Chemical reactions thatlead to a bond formation include, for example, nucleophilic andelectrophilic substitutions, nucleophilic and electrophilic additionreactions, alkylations, addition-elimination reactions, cycloadditionreactions, rearrangement reactions and any other known organic reactionsthat involve a functional group, as well as combinations thereof. Thereactive group may optionally comprise a non-reactive portion (e.g., analkyl) which may serve, for example, to attach a reactive portion of thereactive group to a linking moiety (e.g., poly(alkylene glycol) oranalog thereof) described herein. The reactive group is preferablyselected so as to enable its conjugation to the lysosomal protein.Exemplary reactive groups include, but are not limited to, carboxylate(e.g., —CO₂H), thiol (—SH), amine (—NH₂), halo, azide (—N₃), isocyanate(—NCO), isothiocyanate (—N═C═S), hydroxy (—OH), carbonyl (e.g.,aldehyde), maleimide, sulfate, phosphate, sulfonyl (e.g. mesyl, tosyl),etc. as well as activated groups, such as N-hydroxysuccinimide (NHS)(e.g. NHS esters), sulfo-N-hydroxysuccinimide, anhydride, acyl halide(—C(═O)-halogen) etc. In some embodiments, the reactive group comprisesa leaving group, such as a leaving group susceptible to nucleophilicsubstitution (e.g., halo, sulfate, phosphate, carboxylate,N-hydroxysuccinimide).

The invention also provides the inventive lysosomal protein orcomposition as a medicament. Further provided is a method of treatmentof a lysosomal storage disease comprising administering a lysosomalprotein composition to a patient, e.g. a mammal. Related thereto, theinvention provides the inventive lysosomal protein for use in thetreatment of a lysosomal storage disease. The patient can be amammalian, especially human or a non-human animal, such as a rodent, adog, cat, horse, cow, camel, pig. Preferably the lysosomal protein isfrom the same species as the patient in order to prevent immunoreactionsagainst the proteins amino acid chain.

Preferably the disease and lysosomal protein are selected from thefollowing table:

disease lysosomal protein Fabry Disease α-Galactosidase A (GLA)Gaucher's Disease β-Glucoceramidase, β-glucosidase (glucocerebrosidase)Alpha-Mannosidesis α-Mannosidase AspartylglucosaminuriaAspartylglucosaminidase Beta-Mannosidosis β-Mannosidase Farber DiseaseAcid Ceremidase Fucosidosis α-Fucosidase GM1-Gangliosidosisβ-Galactosidase, β-Hexosaminidase activator protein Krabbe DiseaseGalactocerebrosidase; Galactoceramidase Lysosomal Acid Lipase lysosomalacid lipase (LAL) (LAL) Deficiency Mucopolysaccharidoses (MPS, TypeI-IX) MPS I α-Iduronidase MPS II Iduronate-2-sulfatase MPS IIIAGlucosamine-N-sulfatase; Heparansulfatsulfamidase (SGSH) MPS IIIBα-N-acetyl-glucosaminidase (NAGLU) MPS IIIC α-glucosaminide-N-acetyltransferase MPS IIID N-Acetygalactosamine-6-sulfatase MPS IVAGalactose-6-sulfate sulfatase MPS IVB β-Galactosidase MPS VIN-Acetygalactosamine-4-sulfatase MPS VII β-Glucoronidase MPS IXHyaluronidase Niemann Pick Disease Sphingomyelinase Pompe Disease (Acid)alpha-1,4-glucosidase Sandhoff Disease β-Hexosaminidase, or its αsubunit Schindler Disease Alpha-N-acetylgalactosaminidase (NAGA);α-Galactosaminidase Tay-Sachs Syndrome β-Hexosaminidase A SialidosisNeuraminidase

The dosis for administration is preferably a dosis of 0.05 to 100 mg/kgbody weight, preferably of 0.1 to 50 mg/kg body weight, especiallypreferred of 0.3 to 10 mg/kg body weight, such as 0.3, 1 or 3 mg/kg bodyweight.

Since there is no cure for lysosomal storage diseases a chronictreatment is required with repeated administrations of the enzymereplacement in regular intervals. Preferably the inventive lysosomalprotein is administered at an interval of 1 to 30 days, preferably of 2to 25 days, more preferred of 3 to 23, or even of 4 to 22 days, of 5 to21 days, of 6 to 20 days, of 7 to 19 days, of 8 to 18 days, of 9 to 17days, of 10 to 16 days, or of 11 to 15 days. Especially preferred are 14day intervals. Administration in such intervals allows steady enzymeactivity in the cells lysosomes, countering protein clearance.

The lysosomal proteins may be administered by any route that leads to afunctional enzyme reaching the vascular system, especially the bloodstream. Preferred is intravenous (i.v.) infusion. Further routes ofadministration include intraperitoneal (i.p.), intramuscular (i.m.) andsubcutaneous (s.c.) administration. I.p., i.m. and s.c. administrationroutes may lead to reduced distribution of the lysosomal protein in thetarget tissue (like heart, kidney, liver and spleen), still sufficientamounts can be administered to these tissue via these routes. These non.i.v. routes, in particular i.p., i.m. and s.c., benefit from betterpatient acceptance and usually the benefit outweighs the reduced targettissue distribution. Furthermore, pharmacokinetic profiles of thenon-i.v. administrations are beneficial as the therapeutic enzyme isavailable in patients plasma over a prolonged time period.

In preferred embodiments, the inventive medical treatment with alysosomal protein of the invention (bryophyte produced) is incombination with a lysosomal protein of the same type and qualitativeenzymatic activity but produced in non-plant, especially mammalian orfungal, cells. The non-plant produced lysosomal protein may havephosphorylated mannose for mannose-6-phosphate receptor (M6PR)recognition and cellular uptake. Also, a lysosomal protein with(artificially) phosphorylated mannose of any source may be used incombination with the inventive lysosomal protein. Such phosphorylated ornon-bryophyte lysosomal proteins are already in use, such as Agalsidasealfa (Replagal®) and Agalsidase beta (Fabrazyme®) in case ofalpha-galactosidase suitable for treatment of Fabry Disease; Alglucerase(Ceredase®), Imiglucerase, Velaglucerase alfa (VPRIV), asβ-glucocerebrosidases, suitable for treatment of Gaucher Disease;Alglucosidase alfa (Myozyme®) suitable for the treatment of PompeDisease; Idursulfase (Elaprase®), a lysosomal enzymeiduronate-2-sulfatase suitable for treatment of Hunter syndrome(MPS-II). Also possible are other plant produced lysosomal proteins tobe used in combination, such as Taliglucerase alfa (Elelyso®), aglucocerebrosidase. The phosphorylated and/or non-bryophyte lysosomalprotein may be cross-linked with the paucimannosidic lysosomal protein.The cross-linked di- or multimer is preferably further PEGylated. Thisimproves stability, half-life and uptake.

The inventive lysosomal protein with paucimannosidic glycosylation canalso be combined with a chaperon, in particular a specific ornon-specific chaperon of the lysosomal protein of the same type andqualitative enzymatic activity. A pharmacologic chaperon of lysosomalproteins is e.g. 1-Deoxygalactonojirimycine (Migalastat). The chaperonis capable to modify re-establish some activity of a dysfunctionalendogenous lysosomal protein in a lysosomal storage disease. Theendogenous lysosomal protein can, and usually is, a phosphorylatedlysosomal protein and can complement receptor interaction of theinventive paucimannosidic lysosomal protein—as described above forcombination therapies for administrations of the phosphorylated ornon-bryophyte protein. Without being limited to a specific therapy, itseems the chaperone can restore or increase enzymatic activity of thelysosomal protein, which has impaired activity due to a mutation causingthe lysosomal storage disease. Especially preferred, Migalastat is usedin combination with an inventive paucimannosidic or bryophyte producedalpha-galactosidase and/or used in the treatment of Fabry Disease.

The combination with phosphorylated or non-bryophyte lysosomal enzymes,especially those with M6PR recognition due to phosphorylation,complements uptake activity of the inventive enzymes, allowing to reachall therapeutically relevant cells with increased efficiency and abroader therapeutic scope of application.

The present invention is further illustrated by the following figuresand examples without being limited to these embodiments of the presentinvention. Any element of the examples can be combined with theinventive concepts as described above.

FIGURES

FIG. 1: Linearized expression cassette for lysosomal protein expression(protein sequence: GLA) expression

FIG. 2: Intermediate and final results of a typical purification ofα-gal A (silver-stained SDS-PAGE)

FIG. 3: Purification of glucocerebrosidase from culture supernatant(Coomassie-stained SDS-PAGE)

FIG. 4: Purification of alpha-glucosidase from culture supernatant.Coomassie-stained SDS-PAGE of concentrated eluate fromConA-chromatography. A) moss-GAA eluate versus alglucosidase alfa(Myozyme). B) moss-GAA eluate versus Molecular Weight Standard.

FIG. 5: In vitro characterization of the enzymes. (a) Enzymepreparations separated in SDS-PAGE and stained with Coomassie Blue.Lanes 1 and 2 are moss-aGal and agalsidase alfa respectively. Lanes 3and 4 are moss-aGal and agalsidase alfa digested with PNGase F. Arrow,α-gal A enzymes after digestion; arrowhead, PNGase F (36 KDa). Proteinstandard and molecular weights are shown on left. (b) Moss-aGal andagalsidase alfa (1 ng each) detected by Western blot using a polyclonalantibody specific to human α-gal A. Representative data from 3independent experiments was shown. (c) Specific α-gal A activities ofenzyme preparations determined using artificial substrate4-MU-α-D-galactopyranoside. Protein concentrations were measured by BCAassay. (d) Stability of the enzymes diluted in buffered human plasma andheated at 37° C. (data are means of triplicates). High-mann:high-mannose aGal; moss-aGal: α-galactosidase from moss; Agal-alfa:agalsidase alfa; Agal-beta: agalsidase beta.

FIG. 6: In vitro uptake study. (a) Intracellular α-gal A activities ofFabry patient's fibroblasts (DMN96.125) after overnight incubation withdifferent enzymes in the presence or absence of 5 mM M6P or 2 mg/mlyeast mannan. (b) Gb₃ immunofluorescence staining shows massivelysosomal accumulation of Gb₃ in untreated Fabry patient's fibroblasts(upper) and significantly decreased Gb₃ in the cells that were treatedwith moss-aGal (lower). (c and d) MR expression in Fabry patient'sfibroblasts and microvascular endothelial cells IMFE1. IMFE1 cells wereMR-positive determined by both western blot (c) and immunofluorescencestaining (d), while the fibroblasts were MR-negative. (e) Intracellularα-gal A activities of IMFE1 cells after overnight incubation withdifferent enzymes in the presence or absence of 5 mM M6P or 2 mg/mlyeast mannan. (f) Uptake rates of different enzymes in IMFE1 cells.Cells were harvested at indicated time points and intracellularactivities were measured. ***P<0.001, moss-aGal vs. high-mann aGal oragalsidase alfa. (g) Western blot analysis of internalized α-gal A inIMFE1 cells after 3 hours incubation with different enzyme preparations.(h) Binding of different enzymes to IMFE1 cell. After 3 hours incubationat 4° C., cell surface-bound enzymes were determined by enzyme assay.The dotted line indicates activity level of mock-treated IMFE1 cells inthis assay (i.e., background level). *P<0.05, ***P<0.001. All the datain graphs are presented as mean±SEM (n=3-4).

FIG. 7: Plasma pharmacokinetics. (a) Plasma clearance of infusedmoss-aGal and agalsidase alfa analyzed by enzyme activities. (b) Westernblot for α-gal A in plasma at 10 min after infusion. (c) α-gal A proteinamounts in plasma at 5 and 10 min after infusion; western blot bandsintensities were analyzed by densitometry. (d) Correlation between α-galA protein amounts and enzymatic activities in plasma at 10 min afterinjection. Data in (a) and (c) are presented as mean±SEM (n=4-5).*P<0.05, **P<0.01.

FIG. 8: Tissue distribution of infused enzymes. Enzyme preparations wereinjected into Fabry mice, and α-gal A activities in the kidney, heart,spleen and liver were measured 2 hours post-injection. (a) Specificactivities in organs. Data are presented as mean±SEM (n=5). *P<0.05,**P<0.01. (b) Activities in whole organs were calculated and data arepresented as % of total activity recovered from 4 organs. (c) α-gal Aprotein in kidney homogenates detected by western blot. Arrow, specificα-gal A band in moss-aGal-injected mice. No detectable specific band wasseen in agalsidase alfa-injected mice. Arrowhead, approximate positionwhere agalsidase alfa band may migrate to (based on findings shown inFIGS. 2b, 4b ).

FIG. 9: Cellular localization of infused moss-aGal and agalsidase alfa.Cellular distribution of infused enzymes in the heart and kidney wasdetermined by immunohistochemistry (n=2). Representative pictures wereshown. (a) Heart. Asterisks indicate the blood vessels withimmunostaining positive cells (most likely endothelial cells), andarrows indicate positive perivascular cells (presumably macrophages).(b) Kidney. Arrows indicate immunostaining positive tubular epithelialcells. Scale bar: 25 μm. Original magnification: 400×.

FIG. 10: Tissue kinetics of infused enzymes. Enzyme preparations wereinjected into Fabry mice, and α-gal A activities in kidney (a), heart(b), spleen (c) and liver (d) were measured at 2, 24, 48 and 96 hourspost-injection. Data are presented as mean±SEM (n=4-5). *P<0.05,**P<0.01, ***P<0.001.

FIG. 11: Efficacy of moss-aGal in clearing accumulated Gb₃ in tissues.Gb₃ contents in kidney (a), heart (b) and liver (c) were analyzed 7 daysafter a single infusion of either moss-aGal or agalsidase alfa atvarious doses. Data are presented as mean±SEM (n=4-5). *P<0.05,***P<0.001. Statistical significance shown on top of each agalsidasealfa-injected group indicates difference between agalsidase alfa and thesame dose of moss-aGal.

FIG. 12-14: Plasma and tissue activities for i.p. (FIG. 12), i.m. (FIG.13) and s.c. (FIG. 14) administration.

EXAMPLES Example 1: Production of Human Alpha-Galactosidase in MossExample 1.1: Expression Strain Construction

The DNA sequence of the human GLA gene (NCBI Reference Sequence:NM_000169.2) coding for alpha-galactosidase A (α-gal A) without nativesignal sequence (SEQ ID NO: 3) was synthesized as a codon-optimizedversion (SEQ ID NO: 4) and sub-cloned into a moss expression vector byGeneArt/Thermo Fisher Scientific (GENEART AG, Regensburg, Germany).Sequences harboring the α-gal A expression construct and aneomycin-resistance conferring gene (npt II) construct were excised aslinear expression cassettes (FIG. 1) from the plasmids using restrictionenzymes.

In order to generate stable α-gal A-producing moss cell lines,protoplasts of a moss double-knockout line devoid of plant specificα-1,3-fucose and β-1,2-xylose residues on its N-glycans (Koprivova etal. (2004) Plant Biotechnol. J. 2, 517-523; Weise et al. (2007) PlantBiotechnol. J. 5(3), 389-401; WO 2004/057002) were transformed with thepurified expression cassettes in a PEG-based transformation procedure(Strepp et al. (1998) Proc. Natl. Acad. Sci. U.S.A 95, 4368).Transformed moss cells were regenerated and selected for resistanceagainst the antibiotic G418. 2000 resistant moss plantlets were screenedin two consecutive rounds for total α-gal A accumulation per biomasswith the best strain becoming the standard production strain.

The linearized expression cassette comprises the following geneticelements: Physcomitrella actin promoter (P Actin) and 5′ UTR, plantsignal peptide (SP), cDNA sequence of human α-gal without native signalsequence (GLA), Physcomitrella tubulin 3′ UTR, Cauliflower mosaic virus35S promoter (P 35S), neomycin phosphotransferase gene (nptII) andCauliflower mosaic virus 35S terminator (T 35S) (FIG. 1). The plantsignal peptide contained the sequence MAFYKISSVFFIFCFFLIALPFHSYA (SEQ IDNO: 5).

The expression strain is a fully regenerated moss plant having theaGal-transgene stably integrated into its genome under the geneticcontrol of moss derived regulatory elements.

Example 1.2: Enzyme Production

The α-gal A production strain was cultivated for 4 weeks (27 d) in a 20L disposable bag (Cellbag 20, GE Healthcare, Germany) placed in a Wave™Reactor Rocker (BioWave 20 SPS, Wave Biotech AG, Switzerland). Thecultivation parameters were: 25-30 rpm rocking rate, 8° angle,SM07-medium (100 mM NaCl, 6.6 mM KCL, 2.0 mM MgSO₄×7H₂O, 1.8 mM KH₂PO₄,20.4 mM Ca(NO₃)₂×4H₂O, 0.05 mM FeNa-EDTA, 4.9 mM MES, 0.1% (w/v)PEG4000, 100.26 μM H₃BO₃, 0.11 μM CoCl₂×6H₂O, 0.1 μM CuSO₄×5H₂O, 5 μMKI, 85.39 μM MnCl₂×4H₂O, 1.03 μM Na₂MoO₄×2H₂O, 0.11 mM NiCl₂×6H₂O, 0.04μM Na₂SeO₃×5H₂O, 0.039 μM Zn-acetate×2H₂O), 25° C., 0.3 L× min⁻¹pressured air supplemented with 2% to 4% CO₂ and illumination at 130 to310 μE× m⁻²× s⁻¹, 24 h light per day, delivered from light panelsequipped with Osram FQ 24W 840 HO, Lumilux Cool White. The medium wasadditionally supplemented with 1000× Nitsch vitamin mixture (Nitschvitamin mixture, Duchefa, Netherlands) according to manufacturer'sinstructions. The pH of the fermentation was controlled automatically atpH5-6 through titration with 0.5M H₂SO₄ and 0.5M NaOH with help ofWAVEPOD I (GE Healthcare) in combination with Pump20 (GE Healthcare).

After the end of cultivation the culture broth underwent the followingthree step filtration cascade to deliver a moss free, clarified sterilefiltrate: 1) moss harvest through cake filtration in customized PPfiltration housing (Grosse et al. (2014) WO 2014/013045 A1) equippedwith Zetaplus (01SP B3002, 3M, Germany), 2) depth filtration through adouble layer Scale-Up Capsule (E0340FSA60SP03A, 3M, Germany and 3) afinal sterile filtration step (Millipore Express™ Plus, 0.22 μm,Millipore, Germany).

Subsequently the sterile filtrate was concentrated and buffer exchangedusing tangential flow filtration (TFF) (Pall Centramate 500S, 30 kDacutoff cellulose membrane). After a series of three chromatographicsteps (Butyl-650M, DEAE, S) isolated α-gal A and high-mann α-gal A,respectively, were concentrated to approx. 0.5 mg/ml transferred intothe formulation buffer and characterized. The enzyme was stored at ≤−65°C. until further use. Results of a typical purification process aredepicted in FIG. 2.

Enzyme Activity Measurements:

α-gal A activity was measured by a fluorimetric assay using 5 mM4-methylumbelliferyl-α-D-galactopyranoside at pH 4.4 in the presence of0.1M N-acetylgalactosamine, a specific inhibitor of α-galactosidase B.Protein concentration was measured using BCA protein assay kit (Pierce)according to the suppliers instructions. The activity was expressed asμmol/mg protein/hour. Results are summarized in FIG. 5 c.

SDS-PAGE Silver-Staining:

Samples were denatured in SDS sample buffer supplemented with reducingagent (Invitrogen) at 95° C. for 5 min. NuPAGE Bis-Tris 4-12% gels(Invitrogen) were used for protein separation. Silver-staining was doneusing SilverQuest™ Staining Kit (Invitrogen) according to suppliersmanual.

Host-Cell-Protein (HCP)-ELISA

To quantify remaining HCP levels in the purified α-gal A a novel HCPELISA was developed (Biogenes GmbH, Germany). In short, a mockfermentation with the respective wild-type was done, media harvested andconcentrated. The concentrated protein solution was used forimmunization of rabbit. Total IgG were used to generate a sandwich ELISAfor HCP quantitation. Results show a typical depletion of HCPsthroughout the purification process by a factor of 10000.

Example 1.4: Summary of Production

Production of moss-aGal was accomplished in a photoautotrophicfermentation process in a 10 L-single-use disposable bag installed on aWave™-rocker. The moss, grown in absence of any antibiotics, secretedmoss-aGal into the purely mineral culture medium. Illumination of theculture bags was from the outside at an average photon flux of 200μmol/m²s. After having reached its final culture density, the moss wasseparated by cake filtration from the medium and the latter wasclarified by a double layer depth filter system and final sterilefiltration. The resulting cleared medium was concentrated and rebufferedby means of tangential flow filtration.

From this concentrated harvest, the enzyme was purified by a three stepchromatographic approach, consecutively using a hydrophobic interaction(HIC)-, an anionexchange (AIE)- and a cationexchange (CIE)-column.Finally, the eluate from the last column was rebuffered and concentratedto 0.5 mg/ml.

The purification scheme provided pure moss-aGal (host cell protein (HCP)level ˜100 ppm, single band on Coomassie SDS-Page, SE-HPLC purity 99%)with a typical yield of 30%.

Example 2: α-Galactosidase A Comparison Example 2.1: Enzyme Productionand Activity Assay

Paucimannosidic moss aGal was obtained as described in example 1. Thisproduction strain was additionally transformed with a knock-outconstruct targeting the sole Physcomitrella patensN-acetylglucosaminyltransferase I gene (Gnt I) to obtain productionstrains for high-mann aGal. To test the effect of increased number ofterminal mannosyl residues on cellular uptake of the enzyme, α-gal A wasalso produced in a strain that was genetically depleted of itsbeta-1,2-N-acetylglucosaminyltransferase (GNT-I) activity. Theknockout-modification results in an incapability of the moss to performany complex-type glycan processing as all later enzymatic steps lacktheir substrate. Hence, alpha-mannosidase I mediated trimming in thecis-Golgi is the last processing-step and therefore all N-glycans ofthis strain are of the high-mannose type. Human alpha-galactosidaseproduced in this strain is referred to as high-mann aGal. Production andpurification followed the same scheme as in example 1.

Mammalian cell produced Agalsidase alfa (Shire, Replagal®) andAgalsidase beta (Genzyme, Fabrazyme®) were obtained for comparativetesting.

Cell pellets or Mouse tissues were lysed in ice-cold 0.2% Triton X-100in saline. Lysates were centrifuged at 14,000 rpm for 15 min at 4° C.,and the supernatants were used for enzyme assay. α-gal A activity wasmeasured by the fluorimetric assay using 5 mM4-methylumbelliferyl-α-D-galactopyranoside at pH 4.4 in the presence of0.1M N-acetylgalactosamine, a specific inhibitor of α-galactosidase B.Protein concentration was measured using BCA protein assay kit (Pierce).The activity was expressed as nmol/mg protein/hour.

Example 2.2: Glycan Analysis

Glycan analysis of moss-aGal and Agalsidase alfa was done by ProtagenProtein Services (Dortmund, Germany) using HILIC-UPLC-MS. In short,N-glycans were released from the protein enzymatically using PNGase F.After cleanup and desalting isolated glycans were labeled using2-aminiobenzamide (2-AB). Labeled glycans were separated on a ACQUITYUPLC BEH Glycan (2.1×100 mm) column using a linear gradient of 78% to55.9% B (buffer A: 100 mM ammoniumformate pH4.5, buffer B: acetonitrile)in 38.5 min at 60° C. with a flow rate of 0.5 ml/min. Signals of elutingglycans were recorded by a fluorescence detector (excitation at 330 nm,emission at 420 nm). The assignment of fluorescence peaks to therespective glycans was done using recorded m/z values (Xevo-QTOF MS,Waters) and MasLynx software (Version 4.1, Waters).

Glycan analysis of high-mann aGal was performed as follows. About 25 μgof a-Gal was reduced (15 mM DTT), carbamidomethylated (55 mMiodoacetamide) and acetone precipitated (ace-tone:aqueous phase 4:1).The pellet was redissolved in 0.1 M ammonium bicarbonate buffer anddigested for 12 h with either trypsin at 37° C. or chymotrypsin (bothsequencing grade proteases, Roche, Mannheim).

About 3 μg of each digest was loaded on a BioBasic C18 column(BioBasic-18, 150×0.32 mm, 5 μm, Thermo Scientific) using 60 mM ammoniumformate buffer as the aqueous solvent. A gradient from 3 to 75%acetonitrile was developed over 25 min at a flow rate of 6 μL/min.Detection was performed with a Waters QTOF Ultima mass spectrometerequipped with the standard ESI source in the positive ion mode. Dataanalysis was performed manually with MassLynx4.0.

TABLE 1 N-glycan analysis results of Moss aGal and comparative enzymesTerminal Relative % or Enzymes Formula Name mannoses abundance Moss aGalHexNAc2 Hex2 methyl-Hex Man3 + Methyl 2 24%  HexNAc2 Hex3 Man3 2 57% (HexNAc2 Hex3) + HexNAc1 Man3 + 1x NAc 1 10%  (HexNAc2 Hex3) + HexNAc2Man3 + 2x NAc 0 4% (HexNAc2 Hex3) + (Hex)_(n) Man5-Man8 3 4%Unidentified 1% High mann (HexNAc2 Hex3) + Hex2 Man5 3 dominant (HexNAc2Hex3) + Hex1 Man4 2 few (HexNAc2 Hex3) + Hex3 Man6 3 very few (HexNAc2Hex3) + Hex4 Man7 3 very few Agalsidase (HexNAc2 Hex3) + Hex3 3 2% alfa(HexNAc2 Hex3) + Hex2 (Replagal ®) (HexNAc2 Hex3) + HexNAc1 2 4% Hex2(HexNAc2 Hex3 Fuc1) + 1 1% HexNAc1 Phosphorylated glycans 0 24%  28diverse complex/hybrid all 0 63%  structures (each between 0.1% and 7%)Unidentified 7%In view of glycan biochemistry, it can be assumed that HexNAc isN-acetylglucosamine and Hex is Mannose. Lines 1 and 2 of moss aGal, Man3and Man3+Methyl represent paucimannosidic glycosylation. Surprisinglythis fraction yielded about 80%. High mann and Agalsidase alfa representcomparative products.

As compared to Agalsidase alfa, moss aGal has a very homogeneousstructure composition, with high batch consistency. High batch to batchconsistency is a desired property to guarantee reproducibility andfunction expectation.

TABLE 2 Glycan homogeneity/batch-to-batch stability Mammalian cellproduct Moss product (Replagal) (moss-aGal) No. of batches analyzed 2 6No. of different N-glycans 38 7 Mean MAD (mean average 48% 1.15%deviation)* *Mean of all MADs within single glycoforms

Example 2.3: In Vitro Thermostability

Enzymes were diluted in plasma obtained from a healthy individual andwere heated at 37° C. for indicated time lengths. To keep neutral pH,HEPES were added to the plasma at final concentration of 20 mM. Afterheating, α-gal A activities were measured.

Example 2.4: In Vitro Characterization

Moss-aGal had very uniform N-glycans with core-type Man₃GlcNAc₂ asdominant structure (FIG. 5). Carbohydrate chains of moss-aGal werealmost exclusively constituted by mannose and GlcNAc, of which ˜85% wasmannose-terminated, ˜10% had both mannose and GlcNAc terminal residues,and ˜4% was GlcNAc-terminated (Table. 1). In comparison, Man₅GlcNAc₂ wasthe most abundant glycan structure in high-mann aGal (Table. 1) withsome small amount of Man₄, Man₆ and Man₇. As expected, there were nophosphorylated glycans in both moss-aGal and high-mann aGal. Agalsidasealfa showed highly heterogeneous glycan structures, of which ˜24% werephosphorylated glycans, ˜7% were mannose-terminated glycans and 63% werediverse structures (Table. 1).

In SDS-PAGE, moss-aGal was detected as a single major band with a fastermobility than agalsidase alfa (FIG. 5a ), reflecting the lowercarbohydrate content in moss-aGal. After removal of N-glycans by PNGaseF, both moss-aGal and agalsidase alfa migrated to the same position(FIG. 5a ). High-mann aGal had similar mobility to that of moss-aGal. Inwestern blot analysis, both moss-aGal and agalsidase alfa were detectedby a polyclonal antibody to human α-gal A (FIG. 5b ). With the sameamount of protein loaded, the intensity of moss-aGal band in westernblot was 2.14±0.58 times (n=3) of that of agalsidase alfa, likely due tothe shorter sugar chains in moss-aGal that might facilitate theaccessibility of the antibody to the epitope(s).

Specific activities of moss-aGal and high-mann aGal were similar tothose of agalsidase alfa and agalsidase beta (FIG. 5c ).

Moss-aGal and high-mannose moss-aGal had almost the same stability withagalsidase alfa or agalsidase beta when diluted in human plasma andheated at 37° C. (FIG. 2d ).

Example 3: Production of Human Glucocerebrosidase in the Moss, Suitablefor the Treatment of Gaucher Disease Example 3.1 Expression StrainConstruction

The cDNA sequence of the human GBA gene (Uniprot identifierP04062-GLCM_HUMAN, NCBI Reference Sequence: NM_000157.3) was synthesizedand subcloned into a moss expression vector by GeneArt™ (Thermo FisherScientific, GENEART AG, Regensburg, Germany). The sequence used (SEQ IDNO: 7) is coding for human GBA (SEQ ID NO: 6) without the nativeannotated signal peptide (SP), which was replaced by a 26 aa plant SP(accurate cleavage is predicted with a score of 0.522 according to theSignalP4.1 web-tool). The GBA sequence was modified in one single base(base position 21 in SEQ ID 7, AAA→AAG) using an alternative codon forthe amino acid lysine to facilitate cloning (avoiding an HindIIIrestriction site). Sequences harboring the GBA expression construct anda neomycin-resistance conferring gene (npt II) construct were excised aslinear expression cassettes from the plasmids using restriction enzymes.

A moss cell line based on a double-knockout line devoid of plantspecific α-1,3-fucose and β-1,2-xylose residues on its N-glycans asdescribed in example 1.1 was used. In order to generate stableglucocerebrosidase-producing moss cell lines, protoplasts from thisglyco-engineered basic cell line were transformed with the purifiedexpression cassettes (FIG. 1) in a PEG-based transformation procedure asdescribed in example 1.1. The linearized expression cassette comprisesthe same genetic elements as described in example 1.1 and FIG. 1, withthe exception of using a human GBA sequence instead of the α-galsequence (GLA). Transformed moss cells were regenerated and selected forresistance against the antibiotic G418. Around 700 resistant mossplantlets were screened in two consecutive rounds for totalglucocerebrosidase accumulation per biomass with the best strainbecoming the standard production strain. Human glucocerebrosidaseproduced in this strain is referred to as moss-GBA.

Example 3.2: Enzyme Production and Characterization

The same conditions and steps as described for example 1.2 and example 2were used for production. The glucocerebrosidase was purified bytangential flow filtration with a 10 kDa cellulose cassette, cationexchange chromatography (CaptoS) for capturing and gel filtration(Sephadex) for polishing. Purified/enriched glucocerebrosidase isanalysed by WesternBlotting, Coomassie/Silver stained SDS Page andenzyme activity assay. Purified enzyme was stored at −20° C. untilfurther use. Purification steps are shown in FIG. 3. Similar highmannose-rich glycosylations were obtained. In a rerun, higher GlucNacterminated glycans were found of the form MGn and GnGn. Treatment withbeta-N-Acetylglucosaminidase restored the high amount of paucimmanosidicglycan form distribution.

Example 3.3: Enzyme Assay

Activity of purified glucocerebrosidase is assessed by in vitro enzymeactivity assay. Glucocerebrosidase was incubated in 60 mM Na-Citrat, 1.3mM EDTA, 0.15% Triton-X100, 0.125% sodium taurocholate, 1 mM DTT, 2 mM4-Nitrophenyl-beta-D-glucopyranoside, pH6 at 37° C. The reaction wasstopped with 1M NaOH and the product formation was measured atspectroscopically at 405 nm.

Example 4: Production of Human Lysosomal Alpha-Glucosidase in the Moss,Suitable for the Treatment of Pompe Disease Example 4.1 ExpressionStrain Construction

The cDNA sequence of the human GAA gene (Uniprot identifier P10253(LYAG_HUMAN), NCBI Reference Sequence: NM_000152.4) was synthesized andsub-cloned into a moss expression vector by GeneArt™ (Thermo FisherScientific, GENEART AG, Regensburg, Germany). The sequence used (SEQ IDNO: 9) is coding for human GAA precursor (SEQ ID NO: 8) without thenative annotated signal peptide (SP), which was replaced by a 26 aaplant SP (accurate cleavage is predicted with a score of 0.847 accordingto the SignalP4.1 web-tool) and a truncated pro-peptide. The GAAsequence was modified in one single base (base position 2484 in SEQ ID9, ACG→ACA) using an alternative codon for the amino acid threonine tofacilitate cloning (avoiding a Pvul restriction site). Sequencesharboring the GAA expression construct and a neomycin-resistanceconferring gene (npt II) construct were excised as linear expressioncassettes from the plasmids using restriction enzymes.

A moss cell line based on a double-knockout line devoid of plantspecific α-1,3-fucose and β-1,2-xylose residues on its N-glycans asdescribed in example 1.1 was used. In order to generate stablealpha-glucosidase-producing moss cell lines, protoplasts from thisglyco-engineered basic cell line were transformed with the purifiedexpression cassettes (FIG. 1) in a PEG-based transformation procedure asdescribed in example 1.1. The linearized expression cassette comprisesthe same genetic elements as described in example 1.1 and FIG. 1, withthe exception of using a human GAA sequence (SEQ ID 9) instead of theα-gal sequence (GLA) and a different moss-promoter. Transformed mosscells were regenerated and selected for resistance against theantibiotic G418. Around 600 resistant moss plantlets were screened intwo consecutive rounds for total alpha-glucosidase precursoraccumulation per biomass with the best strain becoming the standardproduction strain. Human lysosomal alpha-glucosidase produced in thisstrain is referred to as moss-GAA.

Example 4.2: Enzyme Production and Characterization

The same conditions and steps as described for example 1.2 and example 2were used for production. The alpha-glucosidase was purified by affinitychromatography using Con A Sepharose 4B. Alpha-glucosidase containingmedium was mixed with the same volume of 50 mM sodium acetate, 1M NaClpH5.2 to adjust for proper binding conditions and loaded onto thechromatography column. Elution was achieved by stepwise increase ofconcentration of α-D-methylglucoside. Purified/enrichedalpha-glucosidase is analysed by WesternBlotting, Coomassie/Silverstained SDS Page and enzyme activity assay. Purified enzyme was storedat 4° C. or −20° C. until further use. SDS-PAGE analysis of enrichedmoss-GAA is shown in FIG. 4. Identity of band containing moss-GAA wasconfirmed by MS-analysis.

Alpha-glucosidase has 7 glycosylation sites, termed G1-G7. Glycoformsfor each site were detected with MS/MS. The most in-tense peak for mostsites (except GS2-GnM with 73%) was found to be that of theGnGn-glycoform. Therefore the enzyme preparation was treated withbeta-N-Acetylglucosaminidase to cleave the terminal GlcNac to convertGnM and GnGn to paucimmanosidic glycans.

Example 5: Mannose Receptor-Mediated Delivery of Moss-Madeα-Galactosidase A Efficiently Corrects Enzyme Deficiency in FabryDisease Example 5.1: In Vitro Uptake Study

Fabry patient-derived skin fibroblasts (DMN96.125) and endothelial cellline (IMFE1) were cultured in 10% FBS in DMEM and EGM-2MV (Lonza)respectively. Both cell lines have very low α-gal A activities, and havelysosomal Gb₃ accumulation that is detectable by immunostaining (Shen etal. (2008) Mol Genet Metab 95:163-168). The cells were incubated withα-gal A preparations (at final concentration of 10 μg/ml) in thepresence or absence of 5 mM M6P or 2 mg/ml yeast mannan for indicatedtime lengths. After that, cells were harvested by trypsin treatment(0.25% trypsin/EDTA, 37° C.) that also eliminates extracellular α-gal A.After washing with PBS, the cell pellets were lysed for enzyme assay orwestern blot.

To test the ability of moss enzymes in degradation of accumulated Gb₃,DNN96.125 cells were incubated with α-gal A preparations (10 μg/ml) for4 days with the medium replaced every 1-2 days. Mock-treated cells wereused as untreated controls. Gb₃ was detected by immunostaining asdescribed below.

Enzyme uptake study was performed in Fabry patients-derived fibroblastswith exogenous enzymes at a final concentration of 10 μg/ml. After 18hours incubation, fibroblasts that were loaded with agalsidase alfa oragalsidase beta had markedly increased intracellular α-gal A activities(116- and 134-fold of activity of untreated cells respectively) (FIG. 6a). Uptake of these enzymes was nearly completely inhibited by M6P andwas partially inhibited by yeast mannan, confirming that this uptake waspredominantly through M6PR. Fibroblasts incubated with moss-aGal orhigh-mann aGal had a significantly lower increment of intracellularα-gal A activities (6.4- and 4.8-fold of untreated cells respectively)(FIG. 6a ). Uptake of both moss-aGal and high-mann aGal was notinhibited by either M6P or mannan. This was consistent with little or noexpression of MR in these cells (FIG. 6c,d ). Despite the low uptake,lysosomal accumulation of Gb₃ in Fabry patient's fibroblasts wassignificantly decreased after treatment with moss-aGal or high-mann aGalfor 4 days (FIG. 6b ), suggesting that moss α-gal A enzymes are able todegrade the accumulated substrates in the lysosomes.

Intravenously infused enzyme in ERT is best taken up by the vascularendothelium, which forms the first barrier between blood and rest of thetissues. Furthermore, endothelial cells are a major disease-relevantcell type in some LSD such as Fabry disease. Therefore, we testedenzymatic uptake in Fabry patient-derived microvascular endothelialcells (IMFE1). IMFE1 cells were MR positive when determined by westernblot and immunostaining (FIG. 6c,d ). After an overnight incubation,moss α-gal A enzymes were efficiently taken up by IMFE1 cells (FIG. 6e). The uptake of moss-aGal or high-mann aGal by IMFE1 cells waspredominantly blocked by yeast mannan (˜60-80% inhibition) and wasinhibited by M6P at a less extent (˜2-10%), suggesting that MR mainlycontributes to this uptake. The uptake of agalsidase alfa or agalsidasebeta by IMFE1 cells was mostly inhibited by M6P (˜75-82%) but also bymannan.

In vitro uptake typically reaches a plateau phase after overnightincubation. To compare uptake rates of different α-gal A preparations ina dynamic phase, IMFE1 cells were incubated with the enzymes (10 μg/ml)for shorter time. Uptake of high-mann aGal and agalsidase alfa wasapproximately linear for up to 3 hours, with significantly higher uptakerate of high-mann aGal than agalsidase alfa (FIG. 6f ). Uptake ofmoss-aGal was remarkably higher than high-mann aGal and agalsidase alfaafter 1-hour incubation, and reached a plateau in 1-3 hours (FIG. 6f ).Similar results were obtained in repeated experiments including one withhigher enzyme amount (40 μg/ml). Western blot further confirmed theseresults at the protein level (FIG. 6g ).

To assess enzyme binding efficiencies, IMFE1 cells were incubated withdifferent enzyme preparations (10 μg/ml) at 4° C. in the presence orabsence of M6P or mannan. Three hours later, cell surface-bound α-gal Awas measured by enzyme activity assay. Under this experimentalcondition, no α-gal A activity above background level was detected(activity of untreated cells) in cells incubated with high-mann aGal oragalsidase alfa (FIG. 6h ). Moss-aGal had significantly higher cellularbinding than high-mann aGal or agalsidase alfa (FIG. 6h ). Binding ofmoss-aGal was significantly blocked by mannan but not by M6P.

These results showed that in an assay system using culturedmicrovascular endothelial cells, which is likely more relevant to invivo ERT than cultured fibroblasts, binding and uptake of moss α-gal Aenzymes are more efficient than agalsidase alfa, and this binding/uptakeoccurs through the MR. These in vitro data also suggested thatmoss-produced enzymes could be suitable for ERT in vivo. Sincebinding/uptake of moss-aGal was more efficient than high-mann aGal, weselected the former for subsequent animal studies.

Example 5.2: In Vitro Binding Study

IMFE1 cells in multi-well plate were incubated with α-gal A enzymes (10μg/ml) at 4° C. in the presence or absence of 5 mM M6P or 2 mg/mlmannan. Culture medium EGM-2MV supplemented with 25 mM HEPES was used.Three hours later, the cells were washed with ice-cold PBS for 4 times,and were directly lysed in 0.2% Triton at 4° C. The lysates were usedfor protein assay and α-gal A enzyme assay.

Example 5.3: SDS-PAGE and Western Blot

Samples were denatured in LDS sample buffer (Invitrogen) with 2.5%2-mercaptoethanol at 70° C. for 10 min. NuPAGE Bis-Tris 4-12% or 10%gels (Invitrogen) were used for protein separation. Western blot wasperformed as described previously (Shen et al. (2008) Biochem BiophysRes Commun 369:1071-1075). Primary antibodies used were rabbitpolyclonal antibody to human α-gal A (Shire Human Genetic Therapies,Cambridge, Mass.), mouse monoclonal antibody to mannose receptor (clone15-2, Abcam, Cambridge, Mass.) and goat polyclonal antibody to GAPDH(Santa Cruz Biotechnology, Santa Cruz, Calif.). The α-gal A proteinlevels were quantified by densitometry using ImageJ software.

Example 5.4: Immunofluorescence

Fluorescence immunostaining was performed as described previously (Shenet al. (2008) Mol Genet Metab 95:163-168). Primary antibodies used weremouse monoclonal antibodies to Gb₃ (Seikagaku, Tokyo, Japan) and mannosereceptor (clone 15-2, Abcam). The cells were counterstained with DAPI.

Example 5.5: Animals and Procedures

Fabry mice were produced by breeding pairs of hemizygous males andhomozygous females. Adult (3-6 months old) female Fabry mice were usedthroughout the study. For each experiment, animals with the same agewere used. For Gb₃ clearance studies, female Fabry mice are more suitedthan male Fabry mice, because male mice have testosterone-induced Gb₃synthesis in kidneys that confounds the effect of the infused enzyme indegradation of accumulated Gb₃. For all the injections, enzymepreparations were diluted in saline to a total volume of 200 μl permouse and were injected into Fabry mice via tail vein.

Example 5.6: Plasma Pharmacokinetics

Enzyme preparations were injected at a dose of 1 mg/kg body weight (n=5each). Blood samples were collected by tail bleed using heparinizedcapillaries at 1, 5, 10, 20 and 30 min after injection. Plasma wasseparated and α-gal A enzyme activity in plasma was measured.

Moss-aGal or agalsidase alfa was injected into Fabry mice via tail-veinat a dose of 1 mg/kg body weight (BW), and plasma clearance was analyzedby an in vitro α-gal A enzyme assay. Moss-aGal was more rapidly clearedfrom circulation than agalsidase alfa (FIG. 7a ). To verify that theshorter plasma half-life of moss-aGal is due to more robust uptake bytissues rather than faster enzyme inactivation (denaturation) in thecirculation, enzymes in mouse plasma were analyzed by western blot (FIG.7b ). According to the higher reactivity of the antibody to moss-aGal(FIG. 5b ), the intensities of moss-aGal bands were corrected by afactor of 2.14. Results revealed that α-gal A protein levels inmoss-aGal-infused mice at 5 and 10 min after infusion were significantlylower than in agalsidase alfa-injected mice (FIG. 7c ). Protein levelsof moss-aGal in plasma at 5 and 10 min were 37% and 28% of that ofagalsidase alfa respectively, which were roughly consistent with theenzyme activity data (specific activities in moss-aGal-injected mouseplasma at these 2 time points were 49% and 28% of that in agalsidasealfa-injected mice). Furthermore, there was a strong correlation betweenprotein levels and enzyme activities in plasma (FIG. 7d ). Together within vitro uptake study findings (FIG. 6f-h ), these data suggested thatintravenously administered moss-aGal is more efficiently taken up byvascular endothelial cells and other cell types in the tissues whencompared to agalsidase alfa.

Example 5.7: Biodistribution

Enzyme preparations were injected at a dose of 1 mg/kg body weight (n=5each). Two hours after injection, mice were perfused with saline (toremove blood), and heart, kidneys, spleen and liver were harvested. Thewhole organs were homogenized, and α-gal A activity was measured. Forkidney, both kidneys were combined and homogenized.

Two hours after intravenous injection of either moss-aGal or agalsidasealfa into Fabry mice (1 mg/kg BW), tissue distribution of each enzymepreparation was assessed. Kidneys from moss-aGal-injected mice hadsignificantly higher enzyme activities than agalsidase alfa (FIG. 8a ).The levels of moss-aGal and agalsidase alfa in the heart and spleen werecomparable (FIG. 8a ). The level of moss-aGal in the liver wassignificantly lower than that of agalsidase alfa (FIG. 8a ). Activitiesper whole organs were calculated and ratios between different organswere compared (FIG. 8b ). Among total recovered activities, 94.9% ofmoss-aGal and 97.5% of agalsidase alfa were delivered to the livers(P<0.05). Kidneys of moss-aGal-injected mice had 1.96% of totalactivity, which is significantly higher (P<0.05) than that in agalsidasealfa-injected mice (0.58%). Western blot analysis confirmed the higheruptake of moss-aGal in the kidney compared to agalsidase alfa (FIG. 8c).

To investigate cellular distribution of the infused enzymes,immunohistochemistry was performed on Fabry mouse tissues 24 hours afterinjection of either moss-aGal or agalsidase alfa at 1 mg/kg BW. Specificsignals displayed granular cytoplasmic pattern, presumably reflectinglysosomal localization of the enzyme. Cellular localization of these 2enzymes in the heart and kidney was essentially identical. In hearts,both moss-aGal and agalsidase alfa were detected in capillaries andperivascular cells but not in myocytes (FIG. 9a ). Specific staining wasonly seen in kidney cortical tubular epithelial cells for either enzyme(FIG. 9b ). These results are consistent with cellular distribution ofagalsidase alfa.

Example 5.8: Immunohistochemistry

Moss-aGal or agalsidase alfa was injected via tail-vein at a dose of 1mg/kg body weight (n=2 each). Heart and kidney were harvested 1 dayafter enzyme infusion. Untreated female Fabry mouse tissues were used asnegative controls. Tissues were fixed in formalin, embedded in paraffin,and 5-micron sections were made. Immunohistochemistry was performed byHistopathology and Tissue Shared Resource in Georgetown University(Washington, D.C.). In brief, after heat-induced epitope retrieval incitrate buffer, sections were treated with 3% hydrogen peroxide and 10%normal goat serum, and were incubated with rabbit polyclonal antibody tohuman α-gal A (Shire). After incubation with HRP-labeled secondaryantibody, signals were detected by DAB chromogen, and the sections werecounterstained with hematoxylin. Signal specificity was verified withcontrol staining, in which the primary antibody incubation was omitted.Compared to light and diffuse non-specific staining in untreatedcontrols, specific signal displayed granular cytoplasmic pattern.

Example 5.9: Tissue Stability

Moss-aGal or agalsidase alfa was injected via tail-vein at a dose of 1mg/kg body weight. At 24, 48 and 96 hours post-injection, mice (n=4-5per group) were perfused and organs were harvested and homogenized asdescribed in Biodistribution above.

Example 5.10: Tissue Kinetics

In vivo kinetics of moss-aGal and agalsidase alfa in various organs wereinvestigated following a single intravenous injection. At 2 and 24 hourspost-injection, kidneys from moss-aGal-injected mice had significantlyhigher enzyme activities compared to agalsidase alfa-injected mice (FIG.10a ). However, activities were similar at 48 and 96 hours (FIG. 10a ).In the heart, there was no significant difference between two forms ofenzymes at 2 and 24 hours; however, activities of moss-aGal were lowerthan agalsidase alfa at 48 and 96 hours post-injection (FIG. 10b ). Incomparison to agalsidase alfa-injected mice, moss-aGal-injected mice hadsimilar level of activities in the spleen, and significantly loweractivities in the liver at all time points analyzed (FIG. 10c,d ). Thehalf-lives of moss-aGal and agalsidase alfa in the kidney and heartranged from 2 to 3 days. Moss-aGal had a ˜25% shorter half-life in bothorgans. The half-life of moss-aGal in the liver was significantlyshorter compared to agalsidase alfa (24 vs. 57 hours). The half-lives ofboth enzyme forms in the spleen were similar (˜30 hours).

Example 5.11: Clearance of Tissue Gb₃

Six months old female Fabry mice were used. Moss-aGal or agalsidase alfawas injected via tail-vein at doses of 0.3, 1 and 3 mg/kg body weight(n=4-5 each). Heart, kidney and liver were harvested 1 week after asingle injection. Age- and sex-matched untreated Fabry and WT mice wereused as controls (n=5). Tissues were homogenized and were subjected toglycosphin-golipids extraction and subsequent analysis of Gb₃ bymass-spectrometry as described previously (Durant et al. (2011) J LipidRes 52:1742-1746). Eight isoforms were analyzed and the results shownare the sum of these isoforms. Gb₃ content was expressed as μg/mg totalprotein.

Efficacies of moss-aGal and agalsidase alfa in degrading accumulated Gb₃were compared at 7 days after a single intravenous injection of eitherenzyme to 6 months old Fabry mice. Three different doses (0.3, 1 and 3mg/kg BW) were tested. Untreated Fabry mice had significantly increasedGb₃ levels in kidney, heart and liver compared to untreated WT controls(FIG. 11a-c ). Both forms of enzymes reduced Gb₃ in these organs in adose-dependent manner (FIG. 11a-c ). Moss-aGal and agalsidase alfa hadcomparable efficacy in clearing Gb₃ in the kidney and heart (FIG. 11a,b), except for a better cardiac Gb₃ clearance of agalsidase alfa at thehighest dose (3 mg/kg). In clearing liver Gb₃, agalsidase alfa was muchmore effective than moss-aGal at doses of 0.3 and 1 mg/kg (FIG. 11c ).At a higher dose (3 mg/kg), these 2 enzymes led to similar liver Gb₃levels.

Example 6: Delivery of Moss-Produced Recombinant Human α-Galactosidase Ato Mouse Model of Fabry Disease Via Non-Intravenous Routes

The purpose of these experiments is to test the potential usefulness ofnon-intravenous routes in delivery of moss αGal to target tissues in themouse model.

Example 6.1: Methods

Moss-aGal as described in example 1 was used in a concentration of 0.69mg/ml. Adult (8-11 months) male Fabry mice were used. Moss aGal wasinjected via intraperitoneal (i.p.), intramuscular (i.m.) orsubcutaneous (s.c.) routes. For the latter two routes, enzyme wasinjected into thigh muscles (both sides) and under the loose skinbetween shoulders, respectively. Doses of 1, 3 or 10 mg/kg body weightwere tested. Blood was collected at 0.5, 1, 2, 4, 6 and 24 hourspost-injection, and organs were dissected at 24 hours. Samples werestored at −80 C until use. Plasma from untreated Fabry mice (n=5) wasused for baseline activity. α-Gal A activities in plasma and tissueswere measured using standard 4MU method.

Spectrometry:

After enzyme reactions, fluorescence intensity of released 4MU wasmeasured using SpectroMax M5 (Molecular Devices). This equipment wasused for analysis of samples from i.p. and i.m. injected mice (and allthe samples we have assayed in recent 5 years). However, becausemechanical problem occurred recently, for s.c. injected mouse samplesthe fluorescence was measured using SpectroMax Paradigm (MolecularDevices). 4MU standard curve in SpectroMax Paradigm showed excellentlinearity, and α-gal A activities of mouse tissues and plasma analyzedwere very close to those previously measured using SpectroMax M5 (testedthe same samples). Therefore, data variation by using 2 differentspectrometries in this study should be very small.

Example 6.2: Results and Discussions

i.p. Route (FIG. 12):

Plasma activities reached peak in 0.5-2 hours after injection, decreasedthereafter and returned to baseline level at 6 hours. Activities weredose-dependent. Activities in heart, kidney, liver and spleen increasedin a dose-dependent manner.

i.m. Route (FIG. 13):

Plasma activities reached peak at 0.5 hour after injection, decreasedrapidly thereafter and returned to baseline level at 4 hours. Activitiesin heart, kidney, liver and spleen increased in a dose-dependent manner.One mouse (#14, with dose of 10 mg/kg) had markedly higher tissueactivities than others in the same group; this sample was removed fromdata analysis.

s.c. Route (FIG. 14):

Plasma activities showed similar pattern as i.m. administration.Overall, tissue activities increased in a dose-dependent manner.However, there was no substantial difference in heart and kidneyactivities between doses of 1 and 3 mg/kg; this may be due to relativelylimited absorption rate of this route. At the dose of 10 mg/kg, tissueactivities showed large variations; 2 out of 5 mice had dramaticallyhigher activities than the rest.

Example 6.3: Comparisons

Enzyme delivery efficiency is in the order of i.p.>s.c. (or similar)i.m.

i.p. Vs. i.v.:

α-Gal A activities in heart, kidney, liver and spleen in i.p. injectedmice (1 mg/kg) were 16%, 49%, 17% and 35% those of i.v. injected mice(data from Tissue Stability study, 1 mg/kg, 24 hours post-injection).

s.c. Vs. i.p./i.v.:

Although s.c. injection led to less enzyme delivery to the tissues thani.p. injection, the ratio of decrement in different organs was notproportional. At dose of 1 mg/kg, α-gal A activities in heart, kidney,liver and spleen in s.c. injected mice were 67%, 43%, 22% and 24% thoseof i.p. injected mice. This suggests s.c. route tends to deliver moreenzyme to heart and kidneys relative to liver and spleen. Similarpattern was seen when compared with i.v. administration. Activities inabove organs of s.c. were 11%, 21%, 4% and 9% of that in i.v. injectedmice.

Non-iv routes are an alternative approach for ERT. i.p. seems a goodmethod. Considering use in human, s.c. may be a good candidate. Althoughtissue amounts are lower than in i.v. administration, sufficient amountscan be administered since only low amounts are needed in tissues. If lowtissue activities (e.g., s.c. vs i.v.) of single administration shouldbe insufficient to degrade accumulated Gb₃ in heart and kidneys,repeated injections can overcome this problem. In summary, the positiveaspects of i.p., i.m. and s.c. administration like improved patientacceptance outweigh the reduced target tissue distribution.

Example 7: Discussion

Depending on the proteins characteristics as well as on its plannedapplication, different expression hosts are chosen. Whereas bulkproteins for industrial and food-/feed applications are mostly expressedin prokaryotic hosts like Escherichia coli, pharmaceutical proteinproduction often relies on expression in higher eukaryotic cells likee.g. CHO-(Chinese-hamster-ovary) or plant cells. The latter choices aremainly based on the fact pharmaceutical proteins, mostly being of humanorigin, require complex posttranslational modifications (PTMs) such ase.g. N-glycosylations. Therefore, parallel recombinant expression of thesame protein in different eukaryotic expression systems yields differentproduct qualities with respect to PTM. For instance in case ofN-glycosylation, mammalian cell expression systems tend to yield a veryheterogeneous product mixture with several tens to hundreds of differentN-glycan species on the same protein product. Plant-based expressionsystems in contrast feature a very homogenous N-glycosylation patternwith only a few (typically below ten) different glycan species presenton the produced protein.

In the case of pharmaceutical protein production, the choice of theproduction system is often triggered by the structural and qualitydemands of the product. The present invention was driven by the need toproduce a recombinant lysosomal protein to treat patients suffering fromLSD. As these patients lack a functional version of this enzyme due toinheritable gene mutation, the recombinant product is used as areplacement by means of regular enzyme replacement therapy (e.g.intravenous infusion). For efficient uptake from the blood stream bybinding to the mannose-receptor on surface of the target cells, theenzyme needs to be decorated with N-glycans bearing terminal mannoseresidues.

In order to produce a version of aGal with mannose-terminated N-glycansin a plant expression system, the routine method would have usedvacuolar targeting of the protein by adding a secretion signal to theN-terminus and a vacuolar targeting signal to the C-terminus. In thisapproach, the secretion signal directs the nascent protein into theendoplasmic reticulum (ER) where it is decorated with precursor-glycans.Following the default secretory pathway, the protein is shipped to theGolgi-apparatus and its glycans will be further trimmed and processed upto a typical complex plant-N-glycan form. These glycans end with twoterminal N-acetylglucosamin (GlcNAc) residues covering both possiblemannose ends of such a glycan.

Exposure of these two mannoses at the second-last positions of the twoglycan arms is then achieved by the second targeting peptide, thevacuolar targeting signal at the C-terminus. This peptide binds tovacuolar sorting receptors in the trans-Golgi-network (TGN) andinitiates targeting of the attached protein to the vacuole. Here,beta-N-Acetylhexosaminidase cleaves off the terminal GlcNAcs and therebyexposes the mannose residues. The resulting glycans are classified as“paucimannosidic” and are typical for plant vacuolar proteins.

The present invention, in contrast, omits the step of incorporating aC-terminal vacuolar signal into the proteins sequence. Therefore, therecombinant product is not sorted to the vacuole in the TGN, but furtherfollows the default secretory pathway. In this approach, trimming of thecomplex N-glycans and the associated exposure of terminal mannoses isnot expected, as paucimannosidic structures are assigned to bevacuole-specific (Castilho & Steinkellner, 2012, Biotechnology Journal,7(9), 1088-1098).

The present invention achieved N-glycan-trimming in bryophytes togenerate a recombinant version of lysosomal proteins with exposedterminal mannoses on its N-glycans without a vacuolar signal. This leadsto a secretory pathway that nonetheless, independent of vacuolarglycol-processing led to a product with high amounts of paucimannosidicglycoproteins in case of lysosomal proteins.

Moss-aGal was efficiently taken up by endothelial cells that express MR,and this uptake was blocked by yeast mannan, a specific inhibitor ofMR-mediated endocytosis. Moss-aGal was not effectively taken up by humanskin fibroblasts which do not express MR. These findings indicate thatuptake of moss-aGal is mediated by MR. By contrast, uptake of agalsidasealfa involved both MR and M6PR. Animal studies revealed that enzymeactivity and storage clearance capacity of moss-aGal in mouse hearts andkidneys are overall comparable to that of agalsidase alfa. These resultssuggest that mannose-terminated enzymes can be as effective asM6P-harboring enzymes in the treatment of Fabry disease and in otherLSDs.

The tested moss-aGal is identical to its human counterpart with respectto protein sequence and -structure. Homogenous and predominantlymannose-terminated N-glycosylation is achieved by expression in acustomized moss strain. Furthermore, for the production of high-mannaGal, GNT-I (N-acetyltransferase-glycosaminyltransferase I) has beenknocked out. Transfer of an N-acetylglucosamine to the nascent glycan bythis enzyme forms an essential substrate for their further processing tocomplex forms. Therefore complex glycan processing is blocked in thisknockout and all glycoforms are of the high-mann type.

Our study showed that moss is a useful platform to express α-gal A andother lysosomal enzymes. In one aspect, moss per se features anoutstandingly homogenous N-glycosylation, i.e. as compared to e.g.mammalian cells their proteins exhibit a drastically reduced number ofglycoforms with a highly reproducible percentual distribution. Withregard to pharmaceutical production this is highly advantageous in caseswhere N-glycan qualities are decisive for the therapeutic efficacy of aprotein.

The uptake of moss-aGal by endothelial cells was much more efficientthan that of agalsidase alfa. This was consistent with the fasterclearance of infused moss-aGal from circulation in vivo. Given thatendothelial cells may play an important role in pathophysiology ofvasculopathy and other manifestations in Fabry disease, effectivedelivery to the endothelial cells is advantageous in preventing andcorrecting disease pathology. Our study also showed that, in spite ofincreased terminal mannose residues, binding/uptake of high-mann aGal toendothelial cells was significantly less efficient than paucimannosidicmoss-aGal. This suggested that MR binding efficiency possibly dependsmore on the conformation of glycans, rather than absolute number ofexposed mannose residues.

By immunohistochemistry, moss-aGal was detected in vascular endotheliumand perivascular cells in the heart, which is overall consistent with MRdistribution pattern. It is known that cardiomyocytes endocytosemannosylated ligands via MR or MR-like receptors. Although the enzymewas not detected in muscle cells, the significantly decreased cardiacGb₃ (˜45% decrement in Fabry mice received 1.0 or 3.0 mg/kg moss-aGal)suggests that small amount of enzyme that is under detection limit ofour immunostaining method might be delivered to cardiomyocytes. In thekidney, moss-aGal was only detected in tubular epithelial cells. Themechanism for this uptake is unclear as renal tubules have not beenreported to express MR. A potential interpretation is that tubular cellsexpress other receptor(s) that mediates endocytosis ofmannose-terminated glycoproteins. The presence of such unidentifiedreceptor(s) that has MR-like binding activity has been reported inmurine spleen and lymph node. Reabsorption of filtered enzymes bytubular cells through megalin-mediated endocytosis is anotherpossibility.

Moss-aGal and agalsidase alfa displayed different tissue distributionswhen analyzed 2 hours after infusion. Relative to agalsidase alfa,targeting of moss-aGal to the kidney was significantly enhanced and thedelivery to the liver was significantly reduced. This distributionpattern of moss-aGal is advantageous, as kidney is one of the mainorgans affected in this disease. In the liver, infused agalsidase alfais delivered to both hepatocytes and sinus lining cells (endotheliumand/or Kupffer cells) presumably through M6PR, asialoglycoproteinreceptors and MR. Most M6PR accessible to infused phosphorylated enzymeis contained in the liver. In contrast, moss-aGal will be preferentiallydelivered to endothelial and Kupffer cells via MR.

The half-life of internalized moss-aGal in the heart and kidney wasshorter than agalsidase alfa. This is likely related to lowercarbohydrate content in moss-aGal that may lead to increasedsusceptibility of the enzyme to proteolytic degradation in thelysosomes. Because of the faster turnover, activity of moss-aGal in thekidney 4 days after infusion was similar to that of agalsidase alfa. Thereduction of Gb₃ storage in the kidney and heart mirrored the residualenzyme activities at 4 days post-injection.

The comparison of moss-aGal and agalsidase alfa can serve as a usefulmodel to study the roles of M6PR and MR in tissue uptake of agalsidasealfa. As mentioned, both M6PR and MR mediate delivery of agalsidase alfain vitro, thus it is difficult to determine which receptor pathway ismore responsible for the biodistribution and for the therapeuticresponse of this enzyme in a certain target organs. Despite markedlydifferent sugar chains, cellular localization of agalsidase alfa andmoss-aGal in the heart and kidney was surprisingly similar. Storageclearance efficacy in these organs was similar as well. In other words,compared to a completely non-phosphorylated enzyme, M6P residues inagalsidase alfa did not lead to a wider distribution and more completeGb₃ clearance as one might expect. These findings suggested that MRpathway might play a more important role than M6PR in targetingagalsidase alfa to the heart and kidney.

The invention claimed is:
 1. A lysosomal protein composition comprisinga plurality of lysosomal proteins that are glycosylated according to aglycosylation pattern, wherein said glycosylation pattern has at least60% paucimannosidic N-glycans (molar %), wherein the paucimannosidicN-glycan comprises the structure of formula 1:

wherein a square represents N-Acetylglucosamine (GlcNAc), a circlerepresents mannose (Man), and a circle with a T represents a terminalmannose, wherein one or more of the GlcNAc or Man subunits may beα1,3-fucosylated, α1,6-fucosylated and/or β1,2-xylosylated.
 2. Thelysosomal protein composition according to claim 1 wherein the lysosomalprotein comprises at least one of α-Galactosidase; α-Mannosidase;Aspartylglucosaminidase; β-Mannosidase; Acid Ceremidase; α-Fucosidase;β-Galactosidase, β-Hexosaminidase activator protein;Galactocerebrosidaseb; Galactoceramidase; lysosomal acid lipase (LAL);α-Iduronidase; Iduronate-2-sulfatase; Glucosamine-N-sulfatase;Heparansulfatsulfamidase (SGSH); α-N-acetyl-glucosaminidase (NAGLU);α-glucosaminide-N-acetyltransferase; N-Acetygalactosamine-6-sulfatase;β-Galactosidase; N-Acetygalactosamine-4-sulfatase; β-Glucoronidase;Neuraminidase; Sphingomyelinase; Sphingomyelin phosphodiesterase; Acidalpha-1,4-glucosidase; β-Hexosaminidase, or its a subunit;Alpha-N-acetylgalactosaminidase (NAGA); α-Galactosaminidase;β-Hexosaminidase A; Galactose-6-sulfate sulfatase; and Hyaluronidase. 3.The lysosomal protein composition according to claim 1, wherein one ormore of the GlcNAc or Man subunits may be α1,6-fucosylated.
 4. Thelysosomal protein composition according to claim 1 wherein theglycosylation pattern has at least 1% N-glycans of the formulaGlcNAc₂-Hex₂-methyl-Hex; and/or wherein the glycosylation patterncomprises the following N-glycans: 0% to 35% -GlcNAc₂-(Man₂methyl-Hex);30% to 80% -GlcNAc₂-Man₃; 0% to 30% -GlcNAc₂-Man₃-GlcNAc; 0% to 15%-GlcNAc₂-Man₃-GlcNAc₂; 0% to 5% -GlcNAc₂-Man₃-Hex₂, 0% to 11%-GlcNAc₂-Man₃-Hex₃; 0% to 10% -GlcNAc₂-Man₃-Hex₄; 0% to 10%-GlcNAc₂-Man₃-Hex₅; wherein all of these compounds together amount to100% or less than 100%, wherein GlcNAc is a N-Acetylglucosamine subunit,Man is a mannose subunit, Hex is a hexose subunit, methyl-Hex is amethylated hexose subunit; with the proviso that-GlcNAc₂-(Man₂methyl-Hex) and -GlcNAc₂-Man₃ together amount to at least45%, (all % are molar %); wherein the GlcNAc at the reducing end of theglycan is not fucosylated in any one of the above N-glycans; wherein aMan at a branching point, is not xylosylated in any one of the aboveN-glycans.
 5. The lysosomal protein composition according to claim 1comprising non-phosphorylated lysosomal proteins.
 6. An in vitro methodof processing a lysosomal protein comprising a complex N-glycan, saidmethod comprising providing the lysosomal protein of claim 1 in a sampleand contacting the sample with a bryophyte HEXO, whereby the bryophyteHEXO enzyme cleaves terminal GlcNAc residues from the lysosomal proteinthereby producing a paucimannosidic N-glycan.
 7. The method of treatmentof a lysosomal storage disease comprising administering a lysosomalprotein composition according to claim
 1. 8. The lysosomal proteincomposition according to claim 2, wherein the α-Galactosidase isselected from α-Galactosidase A (GLA); ß-Glucoceramidase, andβ-glucosidase (glucocerebrosidase).
 9. The lysosomal protein compositionaccording to claim 4, wherein the glycosylation pattern furthercomprises at least one of the following N-glycans: 1% to 30%-GlcNAc₂-(Man₂methyl-Hex); 40% to 70% -GlcNAc₂-Man₃; 4% to 22%,-GlcNAc₂-Man₃-GlcNAc; 2% to 12% -GlcNAc₂-Man₃-GlcNAc₂; 0% to 3%-GlcNAc₂-Man₃-Hex₂, 1% to 8% -GlcNAc₂-Man₃-Hex₃; 1% to 7%-GlcNAc₂-Man₃-Hex₄; 1% to 7% -GlcNAc₂-Man₃-Hex₅.
 10. The lysosomalprotein composition according to claim 4, wherein methyl-Hex is 2-0methyl hexose.
 11. The lysosomal protein composition according to claim4, wherein Hex is Man.
 12. The lysosomal protein composition accordingto claim 1, wherein the glycosylation pattern has at least 45%paucimannosidic N-glycans (molar %) that are not α1,3-fucosylated andthat are not β1,2-xylosylated.
 13. The in vitro method of processing alysosomal protein comprising a complex N-glycan according to claim 6,wherein said bryophyte HEXO is an HEXO3 enzyme.
 14. The method oftreatment of a lysosomal storage disease according to claim 7 whereinthe disease and lysosomal protein are selected from the following table:Disease lysosomal protein Fabry Disease α-Galactosidase A (GLA)Gaucher's Disease β-Glucoceramidase, β-glucosidase (glucocerebrosidase)Alpha-Mannosidosis α-Mannosidase AspartylglucosaminuriaAspartylglucosaminidase Beta-Mannosidosis β-Mannosidase Farber DiseaseAcid Ceremidase Fucosidosis α-Fucosidase GM1-Gangliosidosisβ-Galactosidase, β-Hexosaminidase activator protein Krabbe DiseaseGalactocerebrosidase; Galactoceramidase Lysosomal Acid Lipase lysosomalacid lipase (LAL) (LAL) Deficiency Mucopolysaccharidoses α-IduronidaseIduronate-2-sulfatase Glucosamine-N-sulfatase; Heparansulfatsulfamidase(SGSH) α-N-acetyl-glucosaminidase (NAGLU)α-glucosaminide-N-acetyltransferase N-Acetygalactosamine-6-sulfataseGalactose-6-sulfate sulfatase β-GalactosidaseN-Acetygalactosamine-4-sulfatase β-Glucoronidase Hyaluronidase NiemannPick Disease Sphingomyelinase Pompe Disease (Acid) alpha-1,4-glucosidaseSandhoff Disease β-Hexosaminidase, or its α subunit Schindler DiseaseAlpha-N-acetylgalactosaminidase (NAGA); α-Galactosaminidase Tay-SachsSyndrome β-Hexosaminidase A Sialidosis Neuraminidase.


15. The lysosomal protein composition according to claim 1, wherein oneor more of the mannose is methylated.